The minimal hormonal requirements for the in-vitro accumulation of alpha-lactalbumin have been investigated in a marsupial, the tammar (Macropus eugenii). Mammary gland explants from 24-day pregnant tammars cultured in medium containing bovine insulin, cortisol and ovine prolactin showed a progressive increase in accumulation of alpha-lactalbumin during 4 days of incubation. No increment was observed if prolactin was omitted from the medium. However, a similar rate of increase was observed after 3 days of culture in medium containing prolactin alone. This induction of alpha-lactalbumin was maximal at a prolactin concentration of approximately 0.02 mg/l, which corresponds to physiological levels during pregnancy and early lactation. The absence of an effect of bovine insulin on tammar explants is not due to a general unresponsiveness to this hormone since insulin-stimulated DNA synthesis and amino acid uptake was evident after 3 days of culture. The inclusion of tri-iodothyronine and raised concentrations of cortisol in culture media have been shown to modulate alpha-lactalbumin synthesis in eutherian mammals but were without effect in the tammar. In addition, increased levels of progesterone did not inhibit the induction of alpha-lactalbumin, confirming an earlier in-vivo study suggesting that progesterone withdrawal may not be the lactogenic trigger in this species. Thus the pregnant tammar is the only species yet described in which alpha-lactalbumin is induced maximally in vitro in response to a single hormone.