A synthetic DNA fragment was constructed to determine the effect of 5' and 3' neighbors of guanine runs on the binding of chemical carcinogens. Determinations were made on the relative intensity of reactivity between aflatoxin B1 or benzo(a)pyrene and methylnitrosourea or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea with various guanine positions in an endlabeled DNA fragment of known sequence. After reaction, the fragments were depurinated to produce strand breaks to allow Maxam and Gilbert sequencing for guanine positions. Relative reaction intensities were compared densitometrically. 3' neighbors exerted greater influence on carcinogen binding than did 5' neighbors, the influence extended only to the adjacent guanine and depended upon the chemical nature of the carcinogen. In addition, the presence of one carcinogen adduct in the guanine run influenced the formation of a subsequent adduct when the DNA was exposed to a second carcinogen, and this effect also depended on the nature of the second carcinogen. The results suggest that DNA adduct formation in the presence of multiple carcinogens is more complicated than an additive mechanism would suggest.