We have studied the relationship between the length of HIV-1 reverse transcriptase (RT)-mediated nucleotide polymerization and inhibitors of these reactions in cell-free RT assays performed in the presence of either of two dideoxynucleoside triphosphates (ddNTPs), i.e. AZTTP or 3TCTP, or nevirapine, a non-nucleoside RT inhibitor. These reactions employed a heterologous RNA template and three DNA oligonucleotide primers, i.e. pAR, dPR and PA, that yielded distinct full-length products of 65, 192 and 376 nt, respectively, in the absence of inhibitor. We now show that the extent of inhibition of RT activity was greatest with use of the PA primer, which normally yielded the longest reaction product, and that lesser degrees of inhibition were noted in the reactions that generated shorter products. For example, at a concentration of 5 microM AZTTP, the extent of inhibition was 75% with the PA primer but only 40% and <10% when reactions were primed by the dPR and pAR primers, respectively. Similar results were obtained when either a mutated form of HIV RT (i.e. M184V), associated with resistance to 3TC, was tested in the presence of 3TCTP or when RT derived from Moloney murine leukemia virus (M-MuLV) was tested in the presence of AZTTP.