The mammalian secreted ribonucleases (RNases) comprise a large family of structurally related proteins displaying considerable sequence variation, and have been used in evolutionary studies. RNase 1 (RNase A) has been assumed to play a role in digestion, while other members have been suggested to contribute to host defence. Using the recently assembled bovine genome sequence, we characterised the complete repertoire of genes present in the RNaseA family locus in cattle, and compared this with the equivalent locus in the human and mouse genomes. Several additions and corrections to the earlier analysis of the RNase locus in the mouse genome are presented. The bovine locus encodes 19 RNases, of which only six have unambiguous equivalent genes in the other two species. Chromosomal mapping and phylogenetic analysis indicate that a number of distinct gene duplication events have occurred in the cattle lineage since divergence from the human and mouse lineages. Substitution analysis suggests that some of these duplicated genes are under evolutionary pressure for purifying selection and may therefore be important to the physiology of cattle. Expression analysis revealed that individual RNases have a wide pattern of expression, including diverse mucosal epithelia and immune-related cells and tissues. These data clarify the full repertoire of bovine RNases and their relationships to those in humans and mice. They also suggest that RNase gene duplication within the bovine lineage accompanied by altered tissue-specific expression has contributed a survival advantage.