The food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) forms adducts to DNA guanine bases at the C-8 position. No other DNA adduction site has been verified for PhIP, nor has any experimental data been collected on the conformation of the PhIP-DNA covalent complex. To determine if multiple PhIP-DNA adduct species exist, or if PhIP-DNA adducts assume multiple conformations, 2-(acetoxyamino)-1-methyl-6-phenylimidazo[4,5-b]-pyridine (N-acetoxy-PhIP) was reacted with calf thymus DNA, followed by an evaluation of the resulting adduct complexes by fluorescence spectroscopy. Approximately 20% of the N-acetoxy-PhIP formed covalent complexes with DNA. Two major and several minor spots were observed by 32P-postlabeling, suggesting a minimum of two major adduct species. UV/vis spectra of the PhIP-modified DNA also showed heterogeneous formation of PhIP-DNA adducts. Fluorescence excitation and emission spectroscopy with or without fluorescence quenching (silver ion and acrylamide) was used to evaluate the number of adducts formed, and the low-resolution conformation of each adduct. Four adduct fluorophores were observed and assigned the nomenclature PAi, where "PA" denotes PhIP Adduct and i = 1-4 in order of fluorescence emission band energies, with 1 the highest and 4 the lowest energy, respectively. Excitation maxima for the adduct fluorophores ranged from 340 to 370 nm, and emission maxima ranged from 390 to 420 nm. The fluorescence from adduct PA1 was quenched by silver but not acrylamide, suggesting a helix-internal configuration. Adduct PA2 fluorescence was strongly enhanced upon silver binding but was not affected by acrylamide, also indicating that this adduct was internal. The fluorescence from adducts PA3 and PA4 was quenched by acrylamide but not silver; thus PA2 and PA3 were tentatively assigned as solvent-accessible. These data are the first suggesting heterogeneous formation of PhIP adducts to intact DNA, but we cannot as yet determine how many chemical species of adduct are formed or if a given species exists in multiple conformations.