Bellenamine, (R)-3,6-diamino-N-(aminomethyl)hexanamide (molecular weight 174), produced by Streptomyces nashvillensis, which has been reported to have weak antibacterial activity and to slightly enhance the immune response, showed potent activity against human immunodeficiency virus type 1 (HIV-1). Its mode of action was investigated. Bellenamine inhibited de novo infection of human T cells with HIV-1, at a 50% effective concentration (EC50) of 0.62 micrograms/ml (3.6 microM). Its 50% cytotoxic concentration (CC50) was over 2000 micrograms/ml (11.5 mM) and thus its cytotoxicity was quite low. When HIV-1-infected cells were treated with bellenamine or glycosylation inhibitors, they produced virus with reduced infectivity, and thus bellenamine inhibited the secondary spread of HIV-1 in vitro similarly to glycosylation inhibitors. However, bellenamine did not change the apparent molecular weights of env or gag proteins, unlike glycosylation inhibitors. Bellenamine showed no significant activity against virus adsorption, reverse transcriptase, viral protease or the glycosylation process. The antiviral mechanism of bellenamine remains to be examined further.