SV40-based infectious virus constructs were used to produce a high copy number of full-length antisense RNA in essentially every cell in a population. Chloramphenicol acetyltransferase (CAT) cDNA was placed in either the sense or antisense orientation relative to the SV40 early promoter in helper-free recombinant virus. RNA synthesized at high levels from the antisense virus was without effect on the expression of a stably-transfected CAT mini-gene controlled by an interferon-inducible promoter in monkey CV1 and large T antigen-expressing tsCOS cells. In double infection experiments the antisense RNA was similarly without effect on expression from CAT cDNA placed in the sense orientation in a second virus vector. No activation of the ppp(A2'p)nA(n greater than or equal to 2) system was observed after interferon treatment in either type of experiment. There was no evidence, therefore, for the formation of double-stranded (ds)RNA. It can be concluded that a large excess of a full-length antisense RNA is not necessarily sufficient to cause inhibition of gene expression even when interferon treatment is used to enhance any effect of dsRNA.