We have employed a cell-free human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) assay to study the effects of non-nucleoside inhibitors of RT (NNRTI) by directly monitoring specific HIV DNA products using a HIV-1 genome-derived template and an oligodeoxynucleotide primer. As previously shown by ourselves and others, nucleoside analog triphosphates, e.g. 3'-azido-3'-deoxythymidine triphosphate and 2',3'-dideoxyadenosine triphosphate, could directly inhibit HIV RT RNA-dependent DNA polymerase activity by causing chain termination, as visualized in a RT reaction that yields specific DNA products. In contrast, each of two NNRTIs, nevirapine and delavirdine, directly inhibited RT activity without causing chain termination effects. We also analyzed interactions between nucleoside analogs and NNRTIs or among NNRTIs by chain elongation/dNTP incorporation and/or steady-state kinetic assays. Combinations of nevirapine with the triphosphates of either the (-)-strand of 2',3'-dideoxy-3'-thiacytidine or 2',3'-dideoxyadenosine yielded additive/synergistic effects on RT activity. However, only an additive effect was observed when combinations of nevirapine and 3'-azido-3'-deoxythymidine triphosphate were employed. Combinations of nevirapine and delavirdine had an antagonistic effect on the inhibition of HIV-1 RT activity.