Rapid in vivo effects of 1,25-dihydroxyvitamin D3 on muscle calcium metabolism have been reported. In vitro studies have shown that exposure of vitamin D-deficient chick soleus muscles to the sterol for 1-10 minutes causes a significant stimulation of tissue 45Ca uptake which can be suppressed by Ca channel blockers. A parallel increase in muscle membrane calmodulin content that could be mimicked by forskolin was observed. Experiments were carried out to obtain information about the mechanism underlying the fast action of 1,25-dihydroxyvitamin D3. Like the sterol, forskolin (10 microns) rapidly increased (+48% at 5 min) soleus muscle 45Ca uptake and its effect could be reversed by nifedipine (50 microns). In agreement with these observations, 1,25-dihydroxyvitamin D3 markedly elevated tissue cAMP levels within 45 seconds to 5 minutes of treatment in a dose-dependent manner (10(-11)-10(-7) M). Moreover, incubation of isolated muscle microsomes with 1,25-dihydroxyvitamin D3 increased adenylate cyclase activity and caused a similar profile of stimulation of protein phosphorylation with [gamma-32P]-ATP as forskolin. Major changes were detected in proteins whose calmodulin binding ability has been previously shown to be increased by 1,25-dihydroxyvitamin D3. In addition, the calmodulin antagonists fluphenazine and compound 48/80 abolished the increase in muscle Ca uptake and membrane calmodulin content produced by the sterol. The results suggest that 1,25-dihydroxyvitamin D3 activates muscle Ca channels through a direct membrane action which involves cAMP-dependent protein phosphorylation and calmodulin binding.