We have reported that amphipathic helical segments in the cytoplasmic domain of the HIV-1 envelope glycoproteins bind to calmodulin (CaM) with high affinity, and inhibit calmodulin-regulated proteins. To investigate the possible role of calmodulin activity in HIV-1 replication, we investigated the anti-HIV activity of various CaM antagonists--trifluoperazine and naphthalenesulfonamide W13 or W7--in HeLa T4 cells, PBMCs, and various T lymphocytic cell lines. The different CaM antagonists were found to inhibit the proliferation of the different cell types to varying extent. Also, the CaM antagonists were found to exert a greater antiproliferative effect on H9/HIV-1IIIB, as compared to uninfected H9 cells, suggesting a deficit of CaM function in HIV-infected cells. The CaM antagonists inhibited virus-induced cell fusion in HeLa T4 cells infected with a recombinant vaccinia virus expressing HIV-1 envelope proteins at threshold concentrations that do not inhibit cell proliferation. The fusion-inhibitory effects of the CaM antagonists were also observed in cocultures of HIV-infected (H9/HIV-1IIIB) and uninfected H9 cells. Under these conditions, the synthesis and surface expression of the viral glycoproteins were not affected, although the kinetics of processing of HIV envelope precursor was delayed. Virus production from both HIV-infected peripheral blood mononuclear cell (PBMC) and MT-2 cell cultures was inhibited by CaM antagonists at concentrations that were inhibitory to cell proliferation. Surprisingly, threshold concentrations of CaM antagonists that do not inhibit cell proliferation were found to enhance virus production from HIV-infected MT-2 cells, but not PBMCs.(ABSTRACT TRUNCATED AT 250 WORDS)