W e have investigated the effects of cystamine on the replication of human immunodeficiency virus (HIV) in human lymphocytes and macrophages, the natural targets of HIV in vivo. Treatment of chronically infected macrophages with cystamine, at a concentration (500 microM) that did not show any cytotoxic or cytostatic effects, strongly decreased (> 80%) HIV-p24 antigen production and completely abolished the production of infectious viral particles. Cystamine does not affect viral transcription, translation or protein processing; indeed, all HIV proteins are present in a pattern similar to that of nontreated cells. Instead, cystamine interferes with the orderly assembly of HIV virions, as shown by electron microscopy analysis, that reveals only defective viral particles in treated cells. Moreover, suppression of HIV replication, due to the inhibition of proviral DNA formation was observed in acutely infected lymphocytes and macrophages pretreated with cystamine. These results show that cystamine potently suppresses HIV replication in human cells by contemporaneously blocking at least two independent steps of the viral life cycle, without affecting cell viability, suggesting that this compound may represent a new possibility towards the treatment of HIV-1 infection.