During DNA synthesis, the binding of human immunodeficiency virus (HIV) reverse transcriptase (RT) to the template-primer precedes its binding to nucleotide triphosphates. The interaction of oligonucleotide DNA with HIV-1 RT was investigated by using a gel retardation assay. Both homodimeric (p66/p66) and heterodimeric (p66/p51) isoforms of HI V-1 RT were capable of binding the DNA oligomers. Thus, all further studies on the interaction of HIV-1 RT with DNA were done with heterodimeric RT. We have studied the conditions for optimal binding. The formation of the RT-DNA complex was primer-independent, and the extent of DNA binding was indistinguishable for both single-stranded and double-stranded DNA (either blunt-ended or recessed). The DNA binding activity of the RT was found to be dependent on oligonucleotide length. HIV-1 RT binds DNA with no apparent sequence specificity. Hence, this enzyme belongs to the sequence nonspecific DNA binding proteins. The interaction was found to be independent of DNA synthesis. The formation of the RT-DNA complex was not influenced by the presence of either template-complementary or noncomplementary dNTPs, indicating that neither DNA polymerization nor binding of the RT to the dNTP affects the stability of the complex. The gel retardation assay was utilized to examine also the effect of various HIV-1 RT inhibitors (i.e., AZT-TP, ddTTP, TIBO, and 3,5,8-trihydroxy-4-quinolone) on the enzyme-DNA interaction. The results indicate differences in the modes of action of these compounds. While there was a complete destabilization of the RT-DNA complex in the presence of 3,5,8-trihydroxy-4-quinolone, the addition of AZT-TP, ddTTP, or TIBO had no apparent effect on the stability of the complex. Most effective anti-HIV compounds are inhibitors of HIV RT; hence the interaction of the enzyme with DNA might constitute a discrete step which can serve as a target for interference by novel specific anti-HIV RT drugs.