Pigment Yellow 74 (PY74) is a monoazo pigment that is used in yellow tattoo inks. The metabolism of PY74 was investigated using rat liver and human liver microsomes and expressed human cytochromes P450 (P450s). Two phase I metabolites were isolated and characterized by mass spectrometry and NMR techniques. One metabolite (PY74-M1) was a ring hydroxylation product of PY74, 2-((2-methoxy-4-nitrophenyl)azo)-N-(2-methoxy-4-hydroxyphenyl)-3-oxobutanamide. The second metabolite (PY74-M2) was identified as 2-((2-hydroxy-4-nitrophenyl)azo)-N-(2-methoxy-4-hydroxyphenyl)-3-oxobutanamide, which is the O-demethylation product of PY74-M1. These metabolites were formed by in vitro incubations of PY74 with 3-methylcholanthrene-induced rat liver microsomes and to a much lesser extent by liver microsomes from untreated or phenobarbital-induced rats. The role for CYP1A in the metabolism of PY74 was confirmed using expressed human P450s. The catalytic ability of the P450s for metabolism of PY74 was CYP 1A2 > CYP 1A1 > CYP 3A4 approximately CYP 1B1 (no activity with CYP 2B6, 2C9, 2D6 or 2E1). The metabolism of PY74-M1 to PY74-M2 was catalyzed only by CYP 1A2 and CYP 1A1 (no activity from CYP 1B1, 2B6, 2C9, 2D6, 2E1, or 3A4). These results demonstrate that the tattoo pigment PY74 is metabolized in vitro by P450 to metabolites that should be available for phase II metabolism and excretion.