In the rat thyroid FRTL-5 cell line calcitriol, the biologically most active of the naturally occurring vitamin D metabolites, attenuates both TSH-stimulated cAMP production and the effects of cAMP. Calcitriol treatment abolishes the upregulation of the TSHR number occurring in cells cultivated in the absence of TSH. In addition, the level of G(i-2)alpha increases, which may further attenuate the transmembrane signaling of TSH and facilitate the effects of IGFs. The effect of cAMP on PKAI stimulation is inhibited by increasing the level of the PKA subunit RIIbeta. Regulation of TSHR, G(i-2)alpha and RIIbeta is associated with altered cell proliferation and differentiation in several cells and tissues. Effects of calcitriol on these proteins indicate how the vitamin D endocrine system may regulate cAMP signaling in both classical and nonclassical target tissues.