Administration of the combination of indinavir-zidovudine-lamivudine has been demonstrated to cause a large fraction of treated patients to have a decline in human immunodeficiency virus type 1 (HIV-1) copy number to below the detectability of sensitive assays. A recent investigation (G. L. Drusano, J. A. Bilello, D. S. Stein, M. Nessly, A. Meibohm, E. A. Emini, P. Deutsch, J. Condra, J. Chodakewitz, and D. J. Holder, J. Infect. Dis. 178:360-367, 1998) demonstrated that the durability of the antiviral effect was affected by combination chemotherapy. Zidovudine-lamivudine-indinavir differed significantly from the combination of zidovudine plus indinavir. We hypothesized that the addition of lamivudine might alter the regimen, producing a synergistic anti-HIV effect. In vitro analysis of drug interaction demonstrated that zidovudine-indinavir interacted additively. The addition of lamivudine in concentrations which suppressed viral replication by 20% or less by itself demonstrated marked increases in the synergy volume, increasing the synergy volume 20-fold with the addition of 320 nM lamivudine (which does not suppress HIV by itself) and 40-fold with the addition of 1,000 nM lamivudine (20% viral inhibition as a single agent). A fully parametric analysis with a newly developed model for three-drug interaction confirmed and extended these observations. The interaction term (alpha(IND,AZT, 3TC)) for all three drugs showed the greatest degree of synergy. This marked synergistic interaction among the three agents may explain some of the clinical results which differentiate this regimen from the double-drug regimen of zidovudine plus indinavir.