Two marsupial casein genes have been isolated from a tammar wallaby (Macropus eugenii) mammary gland cDNA library. Comparisons of the tammar alpha- and beta-casein genes with their eutherian homologous reveal extensive divergence at the levels of nucleotide and amino acid sequences. Regions of similarity between the tammar and eutherian Ca(2+)-sensitive caseins are restricted to the major phosphorylation sites and the signal peptides. Quantification of casein mRNA levels in hormone-stimulated mammary gland explants from tammars in late pregnancy suggests that maximal induction of the beta-casein gene is dependent upon prolactin and insulin, while maximal induction of the alpha-casein gene is dependent upon prolactin, insulin and cortisol. These results are in contrast to earlier studies which show that the maximal induction of a putative 19 kDa casein, alpha-lactalbumin and beta-lactoglobulin in the tammar mammary gland is dependent upon prolactin alone. The expression of the latter three genes is not modulated by other hormones known to play a role in the in-vitro initiation of lactation in eutherians.