A transient expression system was used to study the role of the adenovirus late and simian virus 40 (SV40) early mRNA leader sequences and adenovirus virus-associated (VA) RNAs in mRNA translation. Hybrid transcription units containing the adenovirus late and SV40 early promoters fused to various coding regions were introduced into monkey COS cells on plasmids containing a SV40 origin of replication. The translational efficiencies of the mRNAs produced from these plasmids were determined after alterations in the viral leader sequences or in the presence of VA RNAs provided by adenovirus infection of the transfected cells or by cotransfection with plasmids containing the VA genes. Efficient translation of mRNA with either adenovirus or SV40 leader sequences is dependent upon the presence of VA RNA. Translational stimulation by VA RNA of mRNAs containing the adenovirus tripartite leader sequences is dramatically reduced if leader exons 2 and 3 are removed or if their orientation is altered. Sequence analysis has indicated a homology between the nontranslated 5' end of SV40 early mRNA and sequences at the border of the 2nd and 3rd tripartite leader exons, which may be responsible for the increased translation of these mRNAs in the presence of VA RNA.