The direct acting mutagenic N²-hydroxylated metabolite of the food mutagen 2-amino-1-methyl-6-phenylimidazo-[4, 5-b]pyridine (PhIP) does not react with DNA. Upon acetylation of the N²-hydroxy-PhIP with acetic anhydride two products could be detected. Mass spectrometric analysis showed that both products were monoacetyl derivatives of N²-hydroxy-PhIP. One of the products did not show any reactivity towards DNA and is probably the N-acetyl derivative of N²-hydroxy-PhIP. The other product which is most likely to be N²-acetoxy-PhIP reacted with DNA and 2'-deoxyguanosine but not with 2'-deoxycytidine, 2'-deoxy-adenosine or 2'-deoxythymidine. The PhIP-2'-deoxyguanosine adduct was purified and characterized by mass spectral, ¹H and [¹³C]NMR analysis, showing that PhIP like the other cooked food mutagen 2-amino-3-memytiniidazo[4, 5-f]quinoline, had reacted with C-8 of guanine forming N(2'-deoxy-guanosin-8-yl) - PhIP. HPLC analysis of enzymatically hydrolyzed calf thymus DNA which had been reacted with N2-acetoxy-PhIP showed one adduct which was chromato-graphically and spectroscopkally identical to N²-(2'-deoxy-guanosin-8-yl)-PhIP. HPLC separation followed by liquid scintillation counting of hydrolyzed liver DNA from a rat dosed with [³H]PhlP showed that radioactivity coehited with the hydrolysis product of the synthetic PhIP-2-deoxyguanosine adduct, indicating that PhIP in vivo also forms an N²-(2'-deoxyguanosin-8-yl)-PhIP adduct.