Recent studies have provided evidence indicating that 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates calcium influx through Ca2+ channels in isolated chick heart tissue and cells. Studies were performed both to evaluate the characteristics of the hormone action in cultured myocytes and to obtain information on the metabolic pathway which mediates its effects. Treatment of 70-80% confluent chick embryo myocyte monolayers with 1,25(OH)2D3 induced a fast (within 3-5 min) stimulation of 45Ca uptake which was dose-dependent, maximum responses (130% above controls) being elicited at a concentration of 10(-10) M. Physiological levels of 25(OH)D3 and 24,25(OH)2D3, and the synthetic analog 1 alpha (OH)D3, had lower activity. Coincident with the rapid changes in Ca uptake, 1,25(OH)2D3 significantly increased cAMP levels. The hormone-induced increase in cAMP was not blocked by nifedipine. Compound SQ 22536, a specific inhibitor of adenylate cyclase, completely suppressed the effects of the sterol on cAMP and Ca uptake. Furthermore, GDP-beta-S inhibited the increase in Ca uptake by 1,25(OH)2D3. These results involve the adenylate cyclase pathway and the participation of G proteins in 1,25(OH)2D3 stimulation of Ca influx in chick heart cells.