Technical Abstract: Multi-species sequence analysis and other genomics based approaches are being used in our studies of the evolution and regulation of milk protein genes. Multi-species sequence analyses were performed on sequences from Bacterial Artificial Chromosome (BAC) clones isolated and sequenced for this purpose, and from various genome-sequencing efforts. These sequences covered the casein gene cluster region or parts of it in 15 mammalian species: human, chimpanzee, macaque, marmoset, galago, mouse, rat, rabbit, shrew, bat, dog, cow, armadillo, elephant and opossum. This and earlier studies indicated that the casein genes are located in a mammalian specific genomic domain. This domain contains besides the casein genes a number of non-casein genes that share evolutionary ancestry, spatial expression patterns (mammary and salivary gland), and functional properties (secreted (phospho)-proteins, involvement in mineral homeostasis, and immune modulation). The presence and structure of orthologous genes in the mammalian species studied was determined. Predicted transcripts were cloned from a number of species. Phylogenetic analyses showed that the divergence of the casein genes is not only due to a high rate of nucleotide substitutions but also to the differential use of exons. Genomic rearrangements were identified that result in deletions of genomic segments containing casein genes, e.g. lack of alpha-s2-like genes in shrew. Overall there is remarkable conservation in this region with regard to the genes present, gene structure, and gene order and orientation despite high divergence at the nucleotide level. These studies also identified a number of non-coding conserved regions that might play a role in gene regulation. These included the upstream beta-casein enhancer, previously identified in human and cow and now shown to be present in most species studied albeit at different positions with respect to the beta-casein promoter. Computational analyses identified patterns of conservation in these regions and the proximal promoters that represent transcription-factor binding sites known to be involved in casein gene expression.