Direct evidence of Mycobacterium abscessus complex (MABSC) biofilm in the human lung has not previously been demonstrated. Biofilms are microcolonies of bacteria, imbedded in extracellular matrix, providing stability and tolerance to antibiotics and the body's innate and adaptive defences [1]. This mode of growth is an inherent feature of chronic infections and is particularly well studied for Pseudomonas aeruginosa and other Gram-negative infections [1], but also some Staphylococcus aureus infections [2]. Mycobacterial infections have also been shown to be capable of biofilm formation, most notably Mycobacterium tuberculosis (tuberculosis), which under the right conditions, can self-assemble into highly organised matrix-encapsulated biofilm [3]. Among the nontuberculous mycobacteria (NTM), Mycobacterium avium complex (MAC) and the rapidly growing mycobacteria, including MABSC, have been shown to grow as biofilms either in vitro or in environmental reservoirs [4, 5], but in vivo conditions have not been studied. MABSC is an emerging threat to patients with cystic fibrosis [6], who become infected at an early age and deteriorate clinically [7] as the persistent infection causes inflammation and tissue damage. We wanted to explore how MABSC grows in the antibiotic-rich, end-stage lungs of patients with cystic fibrosis. The aim was to describe the localisation and growth patterns of MABSC in vivo from freshly explanted lungs of patients with cystic fibrosis and a history of MASBC. We simultaneously performed histological and mycobacterial sampling from the same areas, from multiple pulmonary sites. Mycobacterial culture was performed by inoculation on at least one solid (Middlebrooke 7H10 or Löwenstein–Jensen slopes; SSI Diagnostica, Hilleroed, Denmark) and in one liquid culture medium (BACTEC 12B or MGIT; Becton Dickinson Microbiology Systems, Sparks, MD, USA), and a reverse hybridisation DNA assay was performed (InnoLiPA; Fujirebio Europe, Brondby, Denmark), as previously described [7]. Culture morphology was determined by direct visual inspection of colonies and control microscopy used Ziehl–Neelsen staining. Concomitant culture for Gram-negative and -positive bacteria was also performed. Patient files were reviewed to determine the clinical course of their end-stage lung disease and their history of other chronic bacterial infections. The NTM collection and clinical data collection was approved by the Committee on Health Research Ethics in the Capital Region of Denmark (Copenhagen, Denmark) (H-3-2012-098). The explanted lungs were collected by the transplantation team, microbiological samples were sent for NTM culture and histological samples were transferred to 4% formaldehyde before further preparation for microscopic investigation. The biopsy material was embedded in paraffin, cut into 4-μm sections and mounted on glass slides. Prior to microscopy, paraffin was removed and the tissue sections were analysed by means of conventional haematoxylin and eosin (H&E) staining, Ziehl–Neelsen staining (acid-fast stain), auramine dye (fluorescent stain), and fluorescent in situ hybridisation (FISH) with peptide nucleic acid (PNA) probes: a uniBac probe (red) and a NTM-specific probe (green) (AdvanDx, Inc., Woburn, MA, USA). We used 4′,6′-diamidino-2-phenylindole (Vector Laboratories) as a counterstain for DNA. Microscopic observations were performed with Zeiss 710 Confocal Laser Scanning Microscope (Leica Microsystems, Mannheim, Germany).