The effects of several vitamin D3 metabolites on TSH secretion were investigated using rat pituitary cells in culture. After a 24-h incubation in Dulbecco's medium, only 25,26-dihydroxyvitamin D3 [25,26(OH)2D3] at 10(-6) M increased the basal TSH secretion (36% above control). The TRH-induced TSH secretion increased dose-dependently with increasing concentrations of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] showing a significant increase at 10(-8) M (41% above control). At 10(-5) M 25,26(OH)2D3 the TRH-stimulated TSH secretion was also increased (63% above control). In medium containing charcoal-treated serum, 1, 24,25-trihydroxyvitamin D3 [1,24,25(OH)3D3] decreased the TRH-induced TSH secretion dose dependently (34% below control at 10(-8) M), while the effects of both 1,25(OH)2D3 and 25,26(OH)2D3 were blunted. Our results indicate that several vitamin D3 metabolites may affect pituitary TSH secretion.