Expression of human immunodeficiency virus (HIV) can be at least partially inhibited by synthetic antisense oligonucleotides (1). Oligodeoxynucleotides complementary to a segment of the viral genome, e.g. the tat-III gene splice acceptor and donor sites, display significant inhibitory effects on viral expression of HIV cultured cells. The effect of exogenous antisense RNA has been rarely investigated because of its rapid degradation in culture. Encapsulation of RNA into liposomes protects against cellular nucleases. Liposomes can be targeted to cells by directly coupled antibodies, or via indirect techniques using protein A to specify the receptor-mediated endocytosis.Here we report that antisense RNA, synthesized using T7 and SP6 RNA polymerase, displayed an anti-HIV-1 effect in the HTLV-IIIB/H9 cell system in vitro. Treatment of HIV-1-infected H9 cells with viral env region antisense RNA encapsulated in liposomes, targeted by antibodies specific for the T cell receptor molecule CD3, almost completely inhibited viral expression. The viral env segment (nucleotides 5585-9153; SstI/SstI) covered the exon II of the HIV-1 tat gene (nucleotides 7925-8230). No anti-HIV activity could be detected with pol RNA (nucleotides 1256-4228; HindIII/Eco-RI) synthesized in either the sense or antisense orientations in similarly targeted liposomes. Env region antisense RNA free in solution, or incorporated in liposomes in the absence of the targeting antibody did not show an inhibitory effect. Western blots showed that antisense env RNA suppressed tat gene expression by approx. 90% and gp160 production by 100%. These results were confirmed by immunoprecipitation studies.We conclude that the antisense env RNA inhibited viral protein production at the translational level and expect that this approach will dominate future studies to develop specific agents in the treatment of viral diseases by gene therapy.