The pig amelogenin gene was isolated from a Lambda genomic library, and a 6.3 kb SalI/XbaI restriction fragment, inclusive of exons 3 through 7, was subcloned into a plasmid vector. DNA sequencing revealed two putative exon 4 sequences. The derived amino acid sequence of exon 4a, KSGRWGARLTAFVSSVQ, had previously been identified in a 190-amino-acid amelogenin isoform by protein sequencing. Exon 4b encoded the peptide DLYLEAIRIDRTAF, which is homologous to exon 4-encoded segments reported for human, mouse, and rat. Oligonucleotides from both of these exons were used to amplify cDNA generated from developing teeth. Amplification products were analyzed by agarose gel electrophoresis, cloned, and characterized by DNA sequencing. Exon 4a was found in transcripts encoding amelogenin isoforms having 190 and 73 amino acids. Exon 4b was found only in apparent splicing intermediates that retained intron 3, but was not detected in any final mRNA transcripts. Pig amelogenin having apparent molecular mass of 23 kD were isolated from the enamel matrix and characterized by mass spectrometry. Two mass values, 18,512.5, and 18,571.2 Da, were measured that match the values predicted for the 162-amino-acid cleavage product of the 173-amino-acid amelogenin, and the 165-amino-acid cleavage product of the 190-amino-acid amelogenin, which includes 17 amino acids encoded by exon 4a. We conclude that the pig amelogenin gene expresses a unique exon 4 that is not homologous to, or evolved from, the exon 4 segment expressed in humans and rodents.