Mixed infections involving equal multiplicities of wild-type simian virus 40 and viable deletion mutant dl861 resulted in decreased cytoplasmic levels of wild-type-derived male mRNA, as well as very low to undetectable levels of mutant-derived late mRNA, as compared with individual infections. The dl861 deletion removes 16 to 25 base pairs from the late leader region. This deletion was shown to be the direct cause of the mixed-infection effect; replacement of the deletion with wild-type sequences restored normal levels of late mRNAs in mixed infections. Other viral functions, e.g., early gene expression and replication, were found to be unaffected by the dl861 deletion. Further examination of the mixed-infection effect showed that the levels of unspliced nuclear precursors of late mRNA, derived from both the mutant and wild-type genomes, were decreased or undetectable, in accord with the cytoplasmic results. Thus, the effect appears to be occurring at the transcriptional level. These data demonstrate a trans-acting effect on late transcription, which is detectable due to the presence of the dl861 mutant in the mixed infection. This finding is indicative of a diffusible factor which exerts a control on simian virus 40 late gene expression at the transcriptional level. A model for positive control of simian virus 40 late gene expression is presented.