The effects of 1,25-dihydroxy-Vitamin D3 on phosphate uptake by cultured chick embryonic muscle cells were investigated. Preincubation of primary myoblast cultures during 4-24 hours with physiological levels of 1,25(OH)2D3 resulted in a significant stimulation of velocity and total capacity of phosphate accumulation by the cells. Maximal responses were obtained at 8 hours of treatment with the sterol. In agreement with previous studies, 25-hydroxy-Vitamin D3 also stimulated myoblast phosphate uptake. 24,25-dihydroxy-Vitamin D3 and vitamin D3 were ineffective. Evidence was obtained indicating that 1,25(OH)2D3 affects the Na(+)-linked component of muscle cell phosphate uptake through a mechanism dependent on "de movo" protein and RNA synthesis.