N‐formyl peptides are cleavage products of bacterial and mitochondrial proteins, and can attract leukocytes to sites of infection or tissue damage. In this study, HL‐60 cell lines expressing the human N‐formyl peptide receptor FPR or its two homologues (FPRL1, FPRL2) were used to determine the receptor selectivity of N‐formylated peptides derived from Listeria monocytogenes or from human mitochondrial proteins. Bacterial peptides were 100‐fold more potent on FPR than on FPRL1, whereas none of them could trigger intracellular signaling through FPRL2. In contrast, N‐formylated hexapeptides corresponding to the N terminus of mitochondrial NADH dehydrogenase subunits 4 (fMLKLIV) and 6 (fMMYALF), and cytochrome c oxidase subunit I (fMFADRW) were equally potent on FPR and FPRL1. They triggered cellular responses with the following order of potency: fMMYALF > fMLKLIV > fMFADRW, with an EC₅₀, in a Fura‐2 calcium mobilization assay, of 10 nM, 44 nM, and 160 nM on FPR‐expressing cells, and 15 nM, 55 nM and 120 nM on FPRL1‐expressing cells. fMMYALF was also a low‐affinity agonist of FPRL2 (EC₅₀ of 1 µM) and was chemotactic for both FPRL1‐ and FPRL2‐expressing cells. We identified novel mitochondrial host‐derived agonists for human N‐formyl‐peptide receptors that might play a role in inflammatory or degenerative processes linked to their stimulation.