The M184V mutation in human immunodeficiency virus (HIV) reverse transcriptase is associated with high-level resistance to both (-)2',3'-dideoxy-3'-thiacytidine (3TC) and (-)2',3'-dideoxy-5-fluoro-3'-thiacytidine as well as low-level resistance to 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, and abacavir. This mutation is also associated with diminished HIV replicative fitness as well as several functional changes in enzyme activity, including diminutions in polymerase processivity, pyrophosphorylysis, and nucleotide primer unblocking. Despite the fact that M184V encodes up to 1,000-fold resistance to 3TC, we asked whether this drug might still display some antiviral effect in regard to viruses containing this mutation. Cell-free assays revealed that high concentrations of 3TC triphosphate (i.e., >100 micro M) could affect chain termination and/or inhibit purified reverse transcriptase containing the M184V substitution. This effect became more pronounced with elongation of reverse transcriptase products. In newly infected cells (i.e., peripheral blood mononuclear cells), we found that the amount of full-length reverse transcriptase product was diminished in the presence of 2 to 10 micro M 3TC, although no decrease in the first product of the reverse transcriptase reaction, i.e., minus strong-stop DNA, was observed. In the presence of two other HIV inhibitors, e.g., nevirapine and indinavir, 3TC exerted additive effects in tissue culture at concentrations only marginally higher than the 50% inhibitory concentration (IC(50)). Reverse transcriptases cloned from clinical isolates harboring M184V in the context of multidrug resistance had similar IC(50) values for 3TC triphosphate compared to reverse transcriptase containing only the M184V mutation. These results suggest that viruses containing M184V can retain a higher degree of sensitivity to 3TC than previously assumed.