Direct synthesis and purification procedures are described for the preparation of adducts derived from the covalent binding of 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene [(+)-anti-BPDE or (+)-BPDE 2] to each of the three guanine residues (trans-N2-dG lesions) in the oligodeoxyribonucleotide d(CTATG1G2G3TATC). The positions of the modified Gs are defined by Maxam-Gilbert sequencing techniques. Six different oligonucleotides with one or two precisely positioned (+)-anti-BPDE residues are identified. The absorbance, circular dichroism and fluorescence characteristics are changed upon formation of duplexes with the complementary strands d(GATACCCATAG). In the doubly-modified oligonucleotides, a broad, excimer-like long wavelength fluorescence emission band is observed with a maximum near 455 nm only if the two (+)-anti-BPDE-modified Gs are adjacent to one another. The covalently attached (+)-anti-BPDE residues decrease the thermodynamic stabilities of the duplexes; their melting points are markedly dependent on the position of the lesions, being highest with the (+)-anti-BPDE residue at G1 (Tm = 40 degrees C, only 2 degrees C lower than in the case of the unmodified oligonucleotide) and lowest when it is situated at G3 (Tm = 29 degrees C). The implications of these and other physical characteristics are discussed. The facile synthesis of these or similar site-specific and stereochemically defined (+)-trans-anti-BPDE-N2-dG lesions in runs of contiguous guanines in oligodeoxyribonucleotides of specified base sequence should be useful for the design of site-directed mutagenesis studies in vitro and in vivo.