A new method for the characterization of pre-mRNA splicing products is presented. In this method RNA molecules are hybridized to an oligodeoxynucleotide complementary to exon sequences upstream of a given 5' splice site, and the RNA strands of the resulting RNA:DNA hybrids are cleaved by RNase H. The cleaved RNAs are then subjected to primer extension using a 32P-labelled primer complementary to exon sequences downstream of an appropriate 3' splice site. Since the primer extension products all terminate at the site of RNase H cleavage, their lengths are indicative of the splice sites utilized. The method simplifies the study of the processing of complex pre-mRNAs by allowing the splicing events between any two exons to be analyzed. We have used this approach to characterize the RNAs generated by expression of the rat tropomyosin 1 (Tm 1) gene in various rat tissues and in cultured cells after transient transfection. The results demonstrate that this method is suitable for the analysis of alternative RNA processing in vivo.