We have recently reported that the in vitro inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcription by inhibitors of reverse transcriptase (RT) occurred most efficiently when the expected DNA products of RT reactions were long (Quan et al. , Nucleic Acids Res. 26:5692-5698, 1998). Here, we have used a quantitative PCR to analyze HIV-1 reverse transcription within acutely infected cells treated with RT inhibitors. We found that levels of minus-strand strong-stop DNA [(-)ssDNA] formed in acutely infected MT2 cells were only slightly reduced if cells were infected with viruses that had been generated in the presence of either azidothymidine or nevirapine (5 microM) and maintained in the presence of this drug throughout the viral adsorption period and thereafter. Control experiments in which virus inoculation of cells was performed at 4 degrees C, followed directly by cell extraction, showed that less than 1% of total (-)ssDNA within acutely infected cells was attributable to its presence within adsorbed virions. In contrast, synthesis of intermediate-length reverse-transcribed DNA products decreased gradually as viral DNA strand elongation took place in the presence of either of these inhibitors. This establishes that nucleoside and nonnucleoside RT inhibitors can exert similar temporal impacts in regard to inhibition of viral DNA synthesis. Generation of full-length viral DNA, as expected, was almost completely blocked in the presence of these antiviral drugs. These results provide insight into the fact that high concentrations of drugs are often needed to yield inhibitory effects in cell-free RT assays performed with short templates, whereas relatively low drug concentrations are often strongly inhibitory in cellular systems.