A retrospective study was conducted to describe risk factors associated with Pneumocystis jiroveci pneumonia (PCP) among HIV-negative patients. During 2002–2004, 50 cases of PCP were identified at Rigshospitalet University Hospital on the basis of histology, PCR and clinical symptoms of PCP. Predisposing conditions included haematological malignancy (72%), inflammatory diseases (14%), solid organ transplantation (6%) and other conditions associated with immunodeficiency (8%). The most common treatment related risk factors were steroid usage (76%) and chemotherapy (72%). In 88% of patients who received steroids, dosage was either lowered or given as pulse-therapy in the 2 weeks preceding the onset of symptoms. Only 1 patient was on PCP prophylaxis at diagnosis and only 8 (16%) patients had previously been given PCP prophylaxis. At diagnosis, 78% of patients were lymphopenic. CD4 counts were available in 17 patients. Only 9 patients (52%) had CD4 count values below 300 cells/µl. The overall mortality attributable to PCP was 14% and was significantly associated with delayed diagnosis and treatment. Among immunocompromized HIV-negative patients, PCP should be particularly suspected in the context of steroid treatment and lymphopenia. Although low CD4 count is associated with a higher risk of PCP, the use of CD4 count as guidance for risk identification or prophylaxis among HIV-negative patients appears insufficient.