Treatment with 1, 25-(OH) 2-D3 (1 ng/ml-25 ng/ml) for periods ranging from 2.5 min. to 60 min. did not alter cyclic AMP levels in bone cells isolated from periosteum-free rat calvaria, or in cells isolated from rat periosteal tissues. 1, 25-(OH) 2-D3 failed to modify the acute increases in cyclic AMP elicited by PTH (10 ng/ml-1 ug/ml). Two separate 1, 25-(OH) 2-D3 preparations, biologically active in other systems, were ineffective under a wide variety of experimental conditions. These results suggest that 1, 25-(OH) 2-D3 is not an acute modulator of cyclic AMP metabolism in PTH-treated and untreated bone cells.