Mechanisms underlying cardiac fibrogenesis in magnesium deficiency are unclear. It was reported earlier from this laboratory that serum from magnesium-deficient rats has a more pronounced stimulatory effect on cell proliferation, net collagen production, and superoxide generation in adult rat cardiac fibroblasts than serum from rats on the control diet. The pro-fibrotic serum factors were, however, not identified. This study tested the hypothesis that circulating angiotensin II may modulate cardiac fibroblast activity in hypomagnesemic rats. Male Sprague-Dawley rats were pair-fed a magnesium-deficient (0.0008% Mg) or -sufficient (0.05%) diet for six days and the effects of serum from these rats on [³H]-thymidine and [³H]-proline incorporation into cardiac fibroblasts from young adult rats were evaluated in the presence of losartan, AT₁ receptor antagonist, and spironolactone, aldosterone antagonist. Losartan and spironolactone markedly attenuated the stimulatory effects in vitro of serum from the magnesium-deficient and control groups but the inhibitory effects were considerably higher in cells exposed to serum from magnesium-deficient animals. Circulating and cardiac tissue levels of angiotensin II were significantly elevated in magnesium-deficient animals (67.6% and 93.1%, respectively, vs. control). Plasma renin activity was 61.9% higher in magnesium-deficient rats but serum ACE activity was comparable in the two groups. Further, preliminary experiments in vivo using enalapril supported a role for angiotensin II in magnesium deficiency. There was no significant difference between the groups in serum aldosterone levels. The findings suggest that circulating angiotensin II and aldosterone may stimulate fibroblast activity and contribute to a fibrogenic response in the heart in magnesium deficiency.