We assessed the effects of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), and the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine (3TC) on reverse transcription in CD4-positive cells by isolating truncated human immunodeficiency virus (HIV) DNA fragments. Jurkat cells were treated with AZT (2 microM), ddI (200 microM), or 3TC (50 microM) prior to infection with HIV. Low-molecular-weight DNA was isolated and amplified by PCR with primer pairs which identify different segments of HIV proviral DNA. We found that the HIV DNA fragments generated from drug-treated, HIV-exposed Jurkat cells were truncated at a ratio of 15:1 [i.e., (-) strong-stop DNA to HIV DNA generated after the first template switch]. Full-length DNA was observed in the case of untreated, HIV-infected cultures. Following nucleoside analog treatment of HIV-exposed Jurkat cells, reverse transcription was terminated only after the synthesis of (-) strong-stop DNA. The nucleoside analogs tested, i.e., AZT, ddI, and 3TC, preferentially chain terminated viral DNA synthesis immediately following the first template switch. The (-) strong-stop HIV DNA was present in AZT-treated and untreated cultures for at least 6 days. We also carried out cell-free reverse transcription/template-switching reactions involving tRNA(Lys3) or a deoxyoligonucleotide as a primer, as a means of studying the selective incorporation of AZT triphosphate into proviral DNA. When reactions were primed with tRNA(Lys3), we found that AZT triphosphate was preferentially incorporated after template switching.