The nitropolycyclic aromatic hydrocarbon 1‐nitropyrene (1‐NP) is an environmental pollutant, a potent bacterial and mammalian mutagen, and a carcinogen. The metabolism of 1‐NP by the human intestinal microbiota was studied using a semicon‐tinuous culture system that simulates the colonic lumen. [3H]‐1‐Nitropyrene was metabolized by the intestinal microbiota to 1‐aminopyrene (1‐AP) and N‐formyl‐1‐amin‐opyrene (FAP) as determined by high‐performance liquid chromatography (HPLC) and mass spectrometry. Twenty‐four hours after the addition of [3H]‐1‐NP, the formy‐lated compound and 1‐AP accounted for 20 and 80% of the total metabolism, respectively. This percentage increased to 66% for FAP after 24 h following 10 d of chronic exposure to unlabeled 1‐NP, suggesting metabolic adaptation to 1‐NP by the microbiota. Both 1‐AP and FAP have been shown to be nonmutagenic towards Salmonella typhimurium TA98, which indicates that the intestinal microflora may potentially detoxify 1‐NP.