In order to define further the mechanisms responsible for tooth amelogenin heterogeneity, seven bovine amelogenin cDNAs were sequenced. On the basis of these sequences, five of the cDNAs could be grouped into one class which differed appreciably in sequence from the second group of two cDNAs. Two overlapping bovine genomic clones were then isolated and shown by sequencing to contain six exons encoding the entire consensus sequence of the class I cDNA. Southern blot analysis of DNA from male and female animals using class I or class II specific oligonucleotide probes suggested that the class I gene sequence was located on the X chromosome while the class II sequence was located on the Y chromosome. Therefore, these results also suggest that the genes on the X and Y chromosomes are both transcribed. Furthermore, the results are consistent with alternative splicing of the class I primary transcript as a potential mechanism for generating amelogenin heterogeneity.