3-Methyladenine-DNA glycosylase from calf thymus recognizes both 3-methyladenine (3-mAde), 7-methylguanine (7-mGua) and 3-methylguanine (3-mGua) residues in calf thymus DNA; the rate of release of 3-mAde is approximately eightfold higher than that for 7-mGua. The best DNA polymer substrates appeared to be those having an A-type helical conformation such as d(A-T)n and d(G-C)n. The Km values for release of 3-mAde and 7-mGua were approximately the same for the above mentioned two substrates whereas the Vmax for excision of 3-mAde was threefold higher than that of 7-mGua. The rate of hydrolysis of 7-mGua residues in d(G-C)n was similar to that found for the excision of 3-mAde in calf thymus DNA. The polymer d(G)n X d(C)m, which possesses a B-type helical conformation, was a poor substrate and the rate of excision here was approximately the same as with calf thymus DNA having the B-type structure. Polyamines greatly influenced the activity and at low concentrations a 50-100% increase in the release of 7-mGua, but not 3-mAde, was observed. With higher concentrations the rate of excision of both bases decreased sharply. The sequence specificity of the DNA glycosylase on naturally occurring DNA was studied using methylated DNA fragments from the plasmid pUC18. The results revealed that some 3-mAde as well as 7-mGua residues were seldom attacked. These 3-mAde residues were positioned either 5' to another Ade residue or in a stretch of pyrimidines, and the 7-mGua residue 3' to another Gua residue. The 3-mAde residue most frequently recognized was situated 3' to another Ade residue, and in the case of 7-mGua it was the central Gua residue in the sequence -G-G-G-.