Antisense RNA can inhibit the expression of messenger RNAs (mRNAs) to which they are complementary by a variety of mechanisms and might provide the basis for antiviral therapies of high selectivity. In a previous study of six retrovirally expressed antisense RNAs targeted to HIV-1IIIB, we found that two significantly reduced HIV-1IIIBreplication. Here we test the degree to which this inhibitory effect tolerates the natural variation found in the nucleotide sequence of different strains of HIV-1. We show that the longer of the two inhibitory antisense RNAs (600 bases) inhibits replication of HIV strains RF, MN and SF2 to at least as great an extent as it does the homologous strain. In contrast, the shorter (71 bases) does not inhibit replication of the heterologous strains. An examination of the predicted positions of the mismatches in the duplexes formed between the IIIB antisense RNAs and the mRNAs of heterologous strains suggests that one requirement of an inhibitory antisense RNA is that it can form a perfect duplex with its target mRNA of at least some 51–64 base-pairs. Although the observations presented here are not definitive proof of this, they are reminiscent of the structural requirements deduced for the double-stranded RNA-mediated induction of interferon and the activation of interferon-induced 2′, 5′-oligo(A) synthetase and protein kinase. We tested the ability of antisense RNA to inhibit HIV replication in Jurkat, CEM, U937 and HeLa-T4 cells. The level of inhibition of HIV-1IIIBreplication varied according to the cell line in which it was expressed, but in all cases was significant.