Evidence to date has failed to show a consistent effect of vitamin D metabolites on PTH secretion. This study was undertaken to assess the possible direct, acute effects of vitamin D metabolites on PTH secretion in vitro. Ethanol has been used in several published studies as the vehicle for vitamin D metabolites. We found that 0.2-1.0% ethanol inhibited PTH release from dispersed bovine parathyroid cells (PTC). Our experiments with vitamin D metabolites used ethanol as a vehicle at a concentration less than 0.1%. When compared to ethanol treatment, 10-100 nM 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 25 and 100 nM 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 100 nM 1,24,25-trihydroxyvitamin D3 (1,24,25(OH)3D3) had no effect on PTH release from PTC incubated for up to 4 h. A combination of 1,25(OH)2D3 and 24,25(OH)2D3 (each 25 or 100 nM) was without effect. Also, 100 nM 1,25(OH)2D3 had no effect on PTH release from either bovine parathyroid gland slices or from parathyroid glands from either vitamin D-replete (+D) or vitamin D-deficient (-D) rats incubated for up to 4 h. The i.v. injection of 1 microgram 1.25(OH)2D3 into -D rats had no effect on either serum PTH or calcium (Ca), either 0.5 or 1.0 h after treatment. Parathyroid glands from -D rats incubated with 0.75 mM Ca secreted more PTH than glands of similar weight from rats given 25 micrograms vitamin D3 3 days earlier, suggesting that vitamin D or a metabolite of vitamin D may modulate the sensitivity of the parathyroid gland to medium Ca. In summary, we found no evidence for a direct, acute effect of vitamin D metabolites on PTH secretion under diverse experimental conditions.