Chinese hamster ovary (CHO) cells in culture were limited for polyamines through the use of alpha-methylornithine (alpha MO), a competitive inhibitor of ornithine decarboxylase. Initial exposure of the cells to the inhibitor caused growth rate and intracellular polyamine content to decline continuously. Reseeding the alpha MO-treated cells into medium containing the inhibitor resulted in steady-state (exponential) growth at cell densities below 5 x 10(3) cells/cm2, at a rate approximately twofold slower than untreated cells. Under these conditions, putrescine and spermidine were undetectable and spermine remained relatively constant at a level approximately half that found in untreated cells. Addition of exogenous putrescine elevated the polyamine content and stimulated the growth of alpha MO-treated cultures. Thus, growth rate correlated with polyamine content in the alpha MO-treated cells. The growth of reseeded, alpha MO-treated cells became nonexponential at a density (5 x 10(3) cells/cm2) far below that at which untreated cells departed from exponential growth (1 x 10(5) cells/cm2). Medium obtained from high density, alpha MO-treated cultures inhibited the growth of cells at low density in the presence of alpha MO. Doubling the concentration of the defined components of conditioned medium did not markedly affect its capacity to inhibit growth. However, dialysis completely not markedly affect its capacity to inhibit growth. However, dialysis completely removed the inhibitory activity from conditioned medium. The results imply that a low molecular weight inhibitor of growth is produced by polyamine-limited cells. This is a variable that must be controlled in studies with polyamine-limited animal cells. Morphological studies indicated that subcellular organelles, including mitochondria, were largely unaffected by treatment with alpha MO. The maintenance of mitochondrial integrity in the presence of alpha MO demonstrates that the swelling of mitochondria observed previously in cells treated with methylglyoxal bis(guanylhydrazone) was not due to polyamine limitation. alpha MO-treated cells did, however, accumulate numerous cytoplasmic vacuoles. The identity of these vacuoles and their relationship to cellular physiology is not yet understood.