Deletion mutants of simian virus 40 defective in late gene functions have been examined for genetic elements that control the biosynthesis of the viral mRNAs. Mutant-specific RNA from infected cells was identified by CH3Hg/(OH)/agarose gel electrophoresis and mapped by the nuclease S1 technique. Altered RNA species, shortened by a size equivalent to the deletion region, can be detected in cells infected with these mutants lacking sequences within the body RNA segments. On the other hand, mutants that lack the 5'-end of the body sequences (a splice junction) fail to accumulate the respective shortened RNA species. In particular, mutant dl-2301 whose deletion includes both the leader and body splice junctions plus the intervening sequences, exhibits a polar effect on a distal gene. Whereas the late region of dl-2301 can be transcribed normally, the mutant defect appears to be associated with little or no accumulation of the mutant-specific late RNA in the infected cells. These results suggest that splice junctions or the intervening sequences, or both, in the viral genome are control signals for post-transcriptional processing of the viral RNA.