The major membrane-associated or transmembrane isoforms of the neural cell adhesion molecule (NCAM) are generated by alternative splicing at the 3' end of the mRNA. Further diversity in NCAM structure is observed in the extracellular region of the polypeptide, where the insertion of additional amino acid residues can result from alternative splicing events occurring at the exon 7-exon 8 and exon 12-exon 13 junctions. Here we report the characterization of tissue-specific patterns of alternative splicing at the exon 12-exon 13 junction by using the polymerase chain reaction. Nine alternatively spliced sequences in rat heart between exon 12 and exon 13 were identified. Each sequence consisted of different combinations of the three small exons (15, 48, and 42 bp in length) and the AAG triplet that make up MSD1, the 108-bp muscle-specific sequence found in human skeletal muscle NCAM (G. Dickson, H.J. Gower, C. H. Barton, H. M. Prentice, V. L. Elsom, S. E. Moore, R. D. Cox, C. Quinn, W. Putt, and F. S. Walsh, Cell 50:1119-1130, 1987). Although the rat equivalent of MSD1 (designated 15+ 48+ 42+ 3+) was detected in all ages of heart examined, it was only one of four or five major splice combinations at any given age. The only alternatively spliced sequence found in the exon 7-exon 8 junction of heart NCAM mRNA was the 30-bp variable alternatively spliced exon previously identified in rat brain. Twenty-seven NCAM forms with distinct sequences were found by analysis of individual NCAM transcripts from postnatal day 1 heart tissue for alternative splicing at the exon 7-exon 8 junction, the exon 12-exon 13 junction and the 3' end. Several combinations of splicing patterns in these three different regions of the gene appeared to be preferentially expressed. The observation that the expression of alternatively spliced forms of NCAM is developmentally regulated suggests a role for NCAM diversity in cardiac development.