We have examined the contribution of 5' leader sequences to expression directed by the simian virus 40 (SV40) late promoter. These studies showed that addition of sequences which contain the late leader 3' splice site to the late promoter led to an increase in the accumulation of mRNA expressed by the promoter. No other sequences within the leader region, between SV40 positions 334 and 560, exhibited a substantial influence on mRNA accumulation. The increase was due, at least in part, to the creation of a spliceable mRNA transcript, since mutation of either the 5' or 3' splice site could attenuate the effect. However, sequences at or near the 3' splice site appeared to play a more important role than did the 5' splice site in bringing about this increase. In many instances, mutation of the 3' splice site also led to the accumulation of extended transcripts, whereas mutation of the 5' splice site did not produce this result in any instance. Analysis of these extended transcripts showed that they retained sequences normally lost upon cleavage and polyadenylation. This finding suggested that mutation of the 3' splice site sequence led to decreases in the efficiency of polyadenylation. We propose that the SV40 late leader sequences positively contribute to expression of the viral late genes by increasing mRNA accumulation via multiple mechanisms, including the enhancement of pre-mRNA polyadenylation efficiency.