The biphasic increase of ornithine decarboxylase activity in mouse mammary gland in organ culture occurs with a hormone-independent first peak and a hormone-dependent second peak. The data presented indicate that a change in the osmolarity of the cellular environment is the major contributing factor for the emergence of the hormone-independent ornithine decarboxylase activity in mammary explants. Thus, incubation of mammary explants for 3 h in a medium diluted 53% with distilled water results in approx. 1000-fold stimulation of enzyme activity over the initial level, whereas a similar dilution of the medium with 0.18 M NaCl or 0.3 M sucrose blocks the increase. The increase in enzyme activity is similarly affected by a reduction of the concentration of NaCl in the culture medium. The hypoosmotic stimulation of ornithine decarboxylase activity appears to be affected at a posttranscriptional level, and is enhanced further by the actions of insulin and prolactin. The hypoosmotic enhancement of ornithine decarboxylase activity produces a large increase in the intracellular concentration of putrescine in mammary explants. However, neither the concentration of spermidine and spermine nor the activity of S-adenosyl-L-methionine decarboxylase is affected. In addition, studies of putrescine transport in mammary explants show that hypotonicity causes an increase in the rate of influx and a decrease in efflux of putrescine with enhancement of intracellular putrescine accumulation. On the other hand, the uptake of spermidine, spermine, amino acids, sugar, and a lipophilic cation, triphenylmethylphosphonium is unaffected. These data suggest a possibility that osmotic alteration in cellular environment causes an incresed need for putrescine in mammary cells, resulting in stimulation of ornithine decarboxylase activity, which may represent a cellular mechanism for maintaining the homeostasis of the intracellular cationic environment.