The interplay of insulin, glucocorticoid and prolactin induces the development of mammary epithelium in vitro by converting non-secretory mammary epithelium into secretory cells. Previous studies on this system indicated that the biosynthesis of spermidine, a naturally-occurring polyamine, may be an important regulatory step in lactogenesis. To elucidate the control mechanism for spermidine biosynthesis, we have studied ornithine decarboxylase, which catalyzes the formation of putrescine, a precursor of seprmidine, and which is considered to be a rate-limiting enzyme in the biosynthesis of spermidine. The data show that the enzyme activity in mammary cells increases biphasically during the culture with three hormones. The first peak of activity occurs independently of the added hormones, whereas the second peak depends on the action of insulin and prolactin. Additional studies indicate possible involvement of cyclic AMP as a stimulant of the first hormone-independent peak of activity. Based on our previous observation that addition of spermidine with insulin and prolactin elicits a marked stimulation of milk protein synthesis in cultured mammary cells, we have studied the uptake and metabolism of exogenous spermidine. The data reveal the existence of a transport system for spermidine in mammary epithelium. Some features of the transport system and subsequent metabolism of the polyamines are described.