1Alpha,25(OH)2D3 is an important negative regulator of parathyroid hormone (PTH) gene transcription. In parathyroid cells, as in other target tissues, 1alpha,25(OH)2D3 is degraded by side chain oxidation by the inducible 24-hydroxylase. We have previously shown that one metabolite of this pathway, 1alpha,23(S),25-(OH)3-24-oxo-D3, potently suppresses PTH synthesis and secretion in cultured bovine parathyroid cells (bPTC). Further examination of the metabolites of 1alpha,25(OH)2D3 in bPTC has revealed another compound that is less polar than 1alpha,25(OH)2D3. By HPLC analysis and mass spectrometry, this metabolite was identified as 1alpha,25(OH)2-3-epi-D3. The activity of this metabol ite on PTH gene transcription was assessed by the steady-state PTH secretion by bPTC after 72-h treatment with concentrations from 10(-11) M to 10(-7) M. 1Alpha,25(OH)2-3-epi-D3 was found to be only slightly, but not significantly, less active than the native 1alpha,25(OH)2D3 in suppressing PTH secretion despite having 30 times lower affinity for the bPTC VDR. Both 1alpha,25(OH)2D3 and 1alpha,25(OH)2-3-epi-D3 maximally suppressed PTH secretion by 50%. Along with 1alpha,25(OH)2-3-epi-D3, the activities of the other two A-ring diastereomers were assessed. 1beta,25(OH)2D3 suppressed PTH only at 10(-7) M with a decrease of only 30%, in good agreement with its low VDR affinity. Surprisingly, 1beta,25(OH)2-3-epi-D3 stimulated PTH secretion by 30-50% at concentrations from 10(-11) M to 10(-8)M and fell to control (untreated) rates at 10(-7) M. The mechanism for this increase in PTH secretion is under investigation. Metabolism studies performed in bPTC cells using high concentrations of 1alpha,25(OH)2D3 substrate showed that in some incubations, the concentration of 1alpha,25(OH)2-3-epi-D3 was even higher than that of the parent substrate 1alpha,25(OH)2D3. This finding indicates a slower rate of metabolism for this diastereomer. Thus, production and accumulation of 1alpha,25(OH)2-3-epi-D3, as a major stable metabolite of 1alpha,25(OH)2D3 in parathyroid glands, may contribute to the prolonged suppressive effect of 1alpha,25(OH)2D3 on PTH gene transcription.