We have characterized a 1.5 kb RNA species in T. thermophila macronuclei previously found in vivo and including intron sequences linked to the 3' exon. This IVS-3' exon RNA could be detected in gels as a discrete molecule only after denaturation of nuclear RNA. After addition of 32P-GTP, as splicing cofactor in a nuclear in vitro system, the IVS-3' exon RNA was labeled at its 5' terminus, as was the by-product of splicing, the excised IVS RNA. The time course of labeling indicates that the IVS-3' exon RNA acts like a reaction intermediate and specifically a kinetic precursor to IVS RNA. Partial nuclease digestions showed that the IVS-3' exon RNA and the IVS RNA have the same 5' terminal sequence. In addition the IVS-3' exon RNA can release the 15-mer oligonucleotide cleaved off during circularization of IVS RNA under conditions of high temperature. Taken together, the structural, functional, and kinetic properties of the IVS-3' exon RNA strongly suggest that it represents a previously postulated in vivo intermediate in the splicing pathway.