The extension of mismatched 3'-termini of DNA was implicated as a major determinant that contributes to the low fidelity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). However, HIV-1 RT exhibits variations in its comparative efficiency to extend different 3'-mismatched base pairs that can result either from the differences in the binding capacity of the enzyme to various mispaired DNAs or from differences in the rate of extension of mispairs by a DNA-bound enzyme. In the current study we have examined the interaction of HIV-1 RT with mispaired template-primer 3'-termini, using a gel retardation assay. HIV-1 RT was found to bind mismatched template-primers with purine-pyrimidine (i.e., A . C) and purine-purine (i.e., A . A and A . G) 3'-terminal mispairs to about the same extent. Hence, HIV-1 RT can be considered (in addition to its other basic features) as a 3'-mismatched DNA binding protein. The stability of the complexes formed between HIV-1 RT and the mismatched template-primers tested seems to be unaffected significantly by neighboring sequences and by the presence of the next complementary dNTP. Thus, the dissimilarities observed previously in extension frequencies in the extension of 3'-terminal mismatches are likely to be due to an inherent property of the HIV-1 RT. The fact that HIV-1 RT binds 3'-mismatch-containing template-primers suggests that unextended mismatched DNA can undergo a rebinding process followed by a 3'-mismatch extension, contributing to further understanding of the low fidelity characteristic of HIV-1 RT. It is possible, therefore, that the interaction of the RT with the DNA may constitute an additional suitable target for the development of specific anti-HIV-1 RT drugs.