We recently purified the calcium-independent processing protease named viral envelope glycoprotein maturase (VEM), that converts human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein precursor gp160 to gp120 and gp41, from the human CD4+ T cell line, Molt-4 clone 8 [Kido, H., Kamoshita, K., Fukutomi, A., and Katunuma, N. (1993) J. Biol. Chem. 268, 13406-13413]. In this report, we deal with the inhibitor specificity and calcium requirement for intracellular gp160 processing in cultured HeLa cells and human CD4+ lymphocytes. Processing of gp160 in these cells infected with recombinant vaccinia virus encoding the gp160 gene was not affected by intracellular calcium depletion induced by the calcium ionophore A23187 and EGTA or by intracellular calcium administration. Processing of gp160 by the purified VEM in vitro was not inhibited by EDTA, EGTA, or the metallo-protease inhibitor phosphoramidon, but was specifically inhibited by a substrate analog, decanoyl-RVKR-chloromethylketone, and the trypsin-type protease inhibitors aprotinin, HI-30, and diisopropyl fluorophosphate (DFP). It was also inhibited by E-64 and thiol reagents. But intracellular gp160 processing was inhibited only by permeable, low molecular mass inhibitors of VEM, such as DFP, E-64, and thiol reagents. Syncytium formation induced by cell surface gp120 was also inhibited by permeable inhibitors of VEM. Taken together, our results indicate that calcium ions may not be essential for intracellular gp160 processing and so HIV-1 gp160 induced by recombinant vaccinia virus may be processed mainly by a protease(s) that does not require calcium ions, such as VEM in these cells.