The recently characterized environmental mutagen and potential carcinogen 1-nitropyrene (NP) is known to bind to DNA in Salmonella typhimurium, and also in anaerobic incubations catalysed by purified xanthine oxidase. In the study the authors show that rat liver S9 supernatant, microsomal and cytosolic subcellular fractions are also able to catalyse the binding of 1-nitropyrene labelled with 14C to calf thymus DNA in vitro. In incubations conducted under air, S9 and microsomes from Charles River CD rats were the most active fractions, and NADPH was required for maximum activity (25-100 pmol NP bound/mg DNA/mg protein in 1 hour). S9 and microsomes had about one-fourth the activity under nitrogen, although less of the activity was NADPH-dependent. Binding in cytosolic incubations was generally low (1 to 5 pmol NP/mg DNA/mg protein in 1 hour), was somewhat enhanced under N2, and was more extensive in the absence of NADPH. Treatment of rats (Harlan Sprague Dawley) with the inducing agents phenobarbital (PB), Aroclor 1254 (A) or 3-methylcholanthrene (3-MC) enhanced NADPH-dependent binding in aerobic S9 (2 to 5-fold) and microsomal (10 to 20-fold) incubations.