Bovine adrenocortical cells of fasciculo-reticulata origin in primary culture actively accumulate polyamines from the extracellular medium in an energy-dependent process. At low extracellular concentration (e.g., 1 microM putrescine), the transport system resulted in a several-hundred-fold concentration of polyamine in the cellular compartment within 1-2 h of incubation. Putrescine uptake appeared to be the sum of a sodium-dependent, saturable process, with an apparent Km of about 10 microM and of a non-saturable, sodium-independent component. By contrast, spermine was taken up by the cells mostly in a sodium-independent manner. Cross-competition experiments suggested that both polyamines were at least partly transported by the same system. Using specific corresponding probes, it was shown that the polyamine uptake was independent of the amino acid transport systems of the A, L and N types known in a number of cell systems. Adrenocortical cell polyamine content is known to be modulated by adrenocorticotropin through induction of ornithine decarboxylase activity. The existence of a specific uptake system in these cells opens the possibility of a more rapid pathway for the regulation of cellular polyamine levels. It remains to be examined whether this polyamine transport system is under hormonal control, and whether this can support the suggestion that polyamines may represent a form of intracellular messengers in the mechanism of hormone action.