We have examined the structures and cellulardistributions of the SV40 late RNAs present in monkey cells at late times after infection. One particular RNA species, spliced at residue 373 (373-RNA), was found to be as abundant as the major late 16S RNAs. This result was unexpected since previous reports showed that the molecular ratio of the 373-spliced 19S RNA to 16S RNA is approximately 0.1 among either cytoplasmic polyadenylated or polysomal viral RNAs. Both sedimentation and electrophoretic analyses indicated that the 373-RNA was approximately 16S to 19S in size. Analysis of the structure of this novel RNA species by S1 mapping and primer extension showed that its 5′ end mapped to the major cap site at nucleotide residue 325 and that nucleotide residue 373 was joined covalently to nucleotide residue 558 as is the case with the majority of the cytoplasmic polyadenylated SV40 late 19S RNAs. However, whereas most SV40 late 16S RNA is polyadenylated and located in the cytoplasm, the majority of 373-RNA was found to lack polyA, exhibit sequence heterogeneity at its 3′ end and be located in the nucleus. The cellular distribution is reversed when the remaining 3′ splice region (around residue 1463) is deleted from this RNA. This implies that the 3` splice region, in the absence of its 5′ splice pair, inhibits the nuclear transport of this 19S spliced RNA, possibly by forming a splicing complex which is an intermediate for the formation of a complete spliceosome.