We have developed a transient expression assay for selection of effective antisense RNAs using episomal replication of plasmids in COS-7 cells, an African green monkey kidney-derived cell line expressing SV40 large T antigen. The transient expression assay was enabled by a liposome-mediated DNA transfection method, by which about 70% of the cells were reproducibly transfected with exogenous DNAs. Plasmids expressing antisense RNAs for the retinoblastoma gene (Rb-1) mRNA and harboring SV40oriwere constructed and introduced into COS-7 cells to examine their inhibitory effect on the accumulation of endogenous Rb protein (pRb). Only the antisense RNA complementary to the 3′-untranslated region (UTR) inRb-1mRNA was expressed stably at high levels for 3 days after the transfection. This antisense RNA reduced by 73% the content of endogenous pRb 70 h after transfection. A similar inhibition was detected in mouse mammary carcinoma cells (FM3A) that were stably transfected with the antisense RNA expressing vector directed to 3′UTR. In contrast, no obvious change in pRb was observed with antisense RNAs complementary to the coding region ofRb-1mRNA. The cellular content of these antisense RNAs was lowered by degradation; thus these RNAs did not affect the levels of pRb in COS-7 and FM3A cells. These results, taken together, suggest that the expression levels and the stability of antisense RNAs are involved in their repressive activity, and our transient expression assay provides a rapid and easy system for evaluation of ectopic antisense RNA activity in COS-7 ce