The D-R cell subline, an ornithine decarboxylase-overproducing variant of L1210 mouse leukemia cells, shows a growth advantage at low osmolality due to its high putrescine content. We tested the ability of spermidine to fulfill the role of putrescine under hyposmotic conditions. Although spermidine (1-30 microM) had no effect on growth under normosmotic conditions (325 mosm/kg), it was strongly inhibitory to D-R cell proliferation at 150 mosm/kg in a concentration-dependent manner. Hypotonic shock greatly increased the rate of spermidine uptake in D-R cells. The increased spermidine content enhanced total putrescine synthesis through a large induction of cytosolic spermidine/spermine N1-acetyltransferase activity but also promoted the excretion of most of the putrescine synthesized by the cells. Delaying the addition of spermidine until 24 h after hypotonic shock resulted in a much sharper decrease in D-R cell viability and strongly depressed polyamine contents. These lethal effects occurred between 8 and 24 h after spermidine addition and followed a dramatic increase in the rate and extent of spermidine accumulation which overrode the metabolic capacity of the N1-acetyltransferase/polyamine oxidase (PAO) pathway. Inhibition of PAO partly reversed the effect of spermidine on growth when the polyamine was added at the time of hypotonic shock, but not 24 h later. Similar experiments performed with alpha-methylspermidine, a metabolically resistant analog, which can completely fulfill cellular requirements for spermidine in normosmotic media, suggested that the lethal effect of a delayed spermidine addition is caused predominantly by excessive accumulation with a minor contribution resulting from stress due to polyamine oxidase activity. In contrast, in hypotonically shocked L1210 cells, spermidine stimulated cell proliferation (albeit less effectively than putrescine), there was no lethal effect of a delayed addition of alpha-methylspermidine, and there was no time-dependent increase in the rate of alpha-methylspermidine uptake. Thus, the spermidine transport system is strongly enhanced by hyposmotic shock in D-R cells, which can result in extensive cell death from overaccumulation of the polyamine and, to a lesser extent, from stress related to the PAO-catalyzed degradation of N1-acetylspermidine. The absence of these effects in parental L1210 cells indicates that the acquisition of an ornithine decarboxylase-overproducing phenotype also involves major modifications in the expression and/or regulation of polyamine transport.