Ouabain, a newly discovered mammalian steroid hormone, has been shown to correlate directly with mean blood pressure. In the present study, the mechanism by which ouabain may act on blood pressure was investigated using primary cultures of bovine vascular smooth muscle cells. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay showed a biphasic effect on cell proliferation: at low concentrations ouabain stimulated cell growth while at higher concentrations it inhibited cell growth, which is similar to angiotensin II, an octapeptide hormone. Confocal laser scanning microscopy assay also indicated a biphasic nature on intracellular free calcium, which was decreased by low concentration of ouabain and increased by higher concentration of ouabain. Simultaneously, an increase of c-myc mRNA expression by low concentration of ouabain was obtained with reverse transcription-polymerase chain reaction (RT-PCR) assay. It is important to note that the low concentrations (10(-15)-10(-12) M) are under the normal circulating levels (2.5-8.0 x 10(-11) M) of endogenous ouabain. These results suggest that ouabain-induced proliferation might be attributed, at least in part, to the decrease of intracellular free calcium and the increase of c-myc mRNA expression, and that may be directly or indirectly involved in the regulation of blood pressure.