It has been shown that supraphysiological concentrations of asparagine and hypoosmotic shock stimulate ornithine decarboxylase activity in cultured cancer cells by increasing the synthesis and the half-life of the enzyme protein. Since extracellular Ca2+ is essential for the action of asparagine and is also important for cell volume regulation in certain cell types, aspects of Ca2+ physiology in asparagine-treated H-35 rat hepatoma cells were investigated. The initial rate of influx of 45Ca increased from 0.25 to 1.04 nmol/min/mg protein immediately after exposure to 10 mM asparagine. With a one-minute lag the efflux rate also increased 2.2-fold over a five minute period. Asparagine did not cause a net-gain in cellular Ca2+ as measured by 45Ca equilibration, nor did it have any effect on the cytosolic free Ca2+ as measured by Fura-2 fluorescence spectroscopy and Fluo-3 fluorescence confocal microscopy.