Phosphate accumulation by muscle in vitro and the effects of vitamin D3 metabolites thereupon were studied in cultures of chick embryo skeletal muscle myoblasts and intact chick soleus muscles. A significant proportion of phosphate accumulation by the cells was Na+-dependent, saturable with respect to phosphate, energy-dependent and inhibited by ouabain and arsenate, in agreement with the operation of a Na+-phosphate cotransport system in the muscle cell plasma membrane as has been described for intestine and kidney. This was further supported by the demonstration of substrate-saturable phosphate uptake in sarcolemma vesicles isolated from chick skeletal muscle. Preincubation of myoblast and soleus muscle cultures with physiological levels of 25-hydroxy-vitamin D3 resulted in a significant stimulation of phosphate accumulation by cultures. 1,25-dihydroxy-vitamin D3 had no effects on the differentiated tissue whereas it markedly increased phosphate accumulation by embryonic muscle cells. In addition, it could be shown that 25-hydroxy-vitamin D3 affects the Na+-linked component of cell phosphate uptake through a mechanism dependent on de novo protein synthesis.