The potential value of HIV-infected human peripheral blood mononuclear cells (PMB) cultured in vitro with phytohemagglutinin (PHA) in the anti-HIV evaluation of nucleoside analogs was examined. 2',3'-Dideoxy-3'-azidothymidine (AZT) exhibited 100-fold higher potency against HIV replication than did 2',3'-dideoxyinosine (ddI) in this system, an observation which does not readily correlate with clinical observations. Studies on metabolism showed that after 12 hr exposure to PHA, cellular AZT was preferentially phosphorylated to its mono-, di-, and triphosphates with 110, 30, and 40-fold increase of the pool sizes, respectively, whereas 2',3'-dideoxycytidine was only moderately phosphorylated, and ddl as well as 2',3'-dideoxyadenosine was poorly phosphorylated. Thymidine kinase activity was found to be the most sensitive to induction among all the cellular kinases regarding stimulation by PHA, and to be responsible for the extreme potency of AZT in this system. Sensitivity of cellular kinases to PHA activation was in the order: thymidine kinase is greater than uridine kinase is greater than deoxycytidine kinase is greater than adenosine kinase. The ratio of ddATP, the active form of ddI, vs. dATP was more than 10-fold higher in resting PBM cells than activated cells. Since the majority of lymphocytes in vivo are in the resting stage and HIV-1 proviral DNA synthesis can be efficiently initiated in resting cells, ddl may exert a disproportionate effect on resting cells rather than activated cells. These data suggest that PHA-stimulated PBM should not be used alone in the screening and evaluation of nucleoside analogues for possible use in the treatment of HIV infections.