The effect of adenovirus VA RNAI on the translation of mRNAs expressing the bacterial chloramphenicol acetyltransferase (CAT) enzyme was studied by a transient expression assay in 293 cells. The CAT activity was determined in extracts prepared from cells transfected with mixtures of plasmids encoding CAT and VA RNA. The results showed that VA RNAI co-transfection resulted in a significant increase in CAT expression from a variety of constructs. Thus, expression of CAT from a SV40 mRNA, a beta-globin mRNA and a chimeric mRNA containing the adenovirus-2 tripartite leader were all stimulated approximately 6-fold by VA RNAI. Based on these results we conclude that the tripartite leader sequence is not required for the VA RNA-mediated stimulation of translation. Our results indicate instead that VA RNAI probably functions as a general enhancer of mRNA translation. A2- to 3-fold stimulation of CAT expression was also obtained following transient expression of HeLa and CV-1 cells. The reduced efficiency was correlated with a 10- to 20-fold lower level of VA RNA expression in HeLa compared with 293 cells. Thus, it is likely that a product from region E1 indirectly enhances the translational efficiency by stimulating VA RNA transcription.