Lamivudine (2′-deoxy-3′-thiacytidine; 3TC) is a dideoxynucleoside analogue that inhibits the replication of human immunodeficiency virus (HIV). We are currently investigating the intracellular metabolism of 3TC to its active triphosphate (3TCTP) in peripheral blood mononuclear cells (PBMC) and a monocytic cell line (U937). Optimal phosphorylation of 3TC was achieved after incubation for 24 hr, with 3TC diphosphate (3TCDP) the predominant metabolite formed, in both cell types investigated. Further studies in PBMCs followed preincubation with the mitogen phytohaemagglutinin (PHA) for 72 hr. This enabled greater detection of phosphates, compared to resting cells. A 3TC concentration of 1 μM was chosen for future interaction studies, allowing good detection of 3TC and phosphates on radiochromatograms whilst being similar to the plasma level found in clinical studies (i.e. 3 μM). With a shift in treatment to combination therapy, it is essential that potential interactions between nucleoside analogues are investigated at the phosphorylation level, as this could affect antiviral activity. Both deoxycytidine (dC) and 2′,3′-dideoxycytidine (ddC) significantly inhibited 3TC phosphorylation (e.g. at dC 100 μM, no 3TCTP was detected in PBMCs; P < 0.001, whereas 66% of control 3TCTP production was observed in U937 cells; P < 0.01). Zidovudine (ZDV) caused a small but significant reduction of 3TC phosphate production in both PBMCs and U937 cells. However, this may be due to toxicity or an effect on endogenous dCTP pools. Neither 2′,3′-dideoxyinosine (ddI) or 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) significantly inhibited 3TC phosphorylation. These results suggest it would be better to coadminister two nucleoside analogues with different activation pathways.