The incubation of Molt-4/HTLV-III cells, human T-lymphotropic virus type III (HTLV-III)/lymphadenopathy-associated virus (LAV)-producer cell line, with more than 0.5 ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA) for 2 to 4 days stimulated virus-induced cell killing which resulted in a high production of HTLV-III/LAV. TPA significantly increased the number of plaque-forming viruses released from the cultures as well as viral RNA content. Interestingly, MT-4 cells freshly infected with HTLV-III/LAV treated for 4 days with 0.125 to 2.0 ng/ml of TPA were found to retain the capacity to grow, because TPA inhibited the induction of the virus-specific antigens and cytopathic effects. In contrast to the situation in infected MT-4 cells in liquid cultures, the addition of 0.125 and 0.25 ng/ml of TPA into agarose medium induced large plaques, suggesting that TPA basically enhanced the production of the virus from one infected MT-4 cell. Taken together, the data suggest that TPA enhances the replication of HTLV/III/LAV. These assay systems are shown to be useful for analyzing the induction or suppression of a virus infection by many drugs and other factors in vitro.