Antisense RNA-mediated inhibition of HIV showed mixed success in previous experiments. In order to elucidate the parameters influencing the efficacy of an antisense RNA approach, retroviral vectors encoding 4.5 kb, 3.5 kb, or 2.5 kb antisense RNA of the gag-pol region of SIV (simian immunodeficiency virus) were constructed and used to transduce a CD4-positive CEM174 cell line. The growth rate of transduced cells was measured, and results showed that antisense RNAs have no detrimental effect on cell growth. Similar levels of antisense RNA expression were observed in all transduced cells by Northern analysis. The transduced cells were challenged with uncloned SIVmac239 at a m.o.i. (multiplicity of infection) of 1.0, 0.1, or 0.01. At a m.o.i. of 1.0 or 0.1, no significant inhibition of viral replication was observed in any antisense construct transduced cells up to 9 days postinfection. At a lower m.o.i. (0.01), viral replication was effectively inhibited in 3.5 kb antisense transduced cells as compared to 4.5 kb and 2.5 kb antisense transduced cells at 15 days postinfection. These data suggest that the size of antisense RNA and the challenge dose play a significant role in achieving effective inhibition.