Polyamine transport was examined in Chinese hamster ovary (CHO) cells because of the unique potential these cells hold for utilizing genetic approaches to study the mechanisms of polyamine transport, its regulation, and its function. Parental (control) CHO cells were shown to contain a polyamine transport system with characteristics consistent with polyamine-uptake properties described in other cell types. Polyamines appear to cross the plasma membrane via an energy-requiring transport system specific for putrescine, spermidine, spermine, and their analogues. A mutant line, CHOMG, selected for resistance to the toxicity of methylglyoxal bis(guanylhydrazone), was shown to lack a functional polyamine transport system. CHOMG cells provided the negative controls necessary to examine the role of polyamine transport in maintenance of intracellular polyamine levels and in the regulation of the polyamine metabolic enzymes. It was found that the repression of ornithine decarboxylase activity by polyamines and the induction of spermidine/spermine-N1 acetyltransferase by polyamine analogues including bis(ethyl)spermine derivatives required the presence of a functional polyamine transport system. The CHO-CHOMG model was also shown to provide a means for establishing the importance of the polyamine transport system in the toxicity of polyamine analogues. The inability of alpha-difluoromethylornithine-treated CHOMG cells to utilize extracellular polyamines to replenish depleted intracellular polyamine levels suggested a means by which polyamine transport-positive cells may be identified. Such a selection procedure will permit the use of CHOMG cells in the isolation of genes encoding proteins involved in polyamine transport.