1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) attenuates the stimulatory effects of cAMP on proliferation and iodide uptake in rat thyroid FRTL-5 cells. This study examines the effects of 1,25-(OH)2D3 on the cAMP-dependent protein kinase (PKA). Cytosol proteins separated by anion exchange chromatography showed increased [3H]cAMP binding activity as well as increased kinase activity in the fractions containing PKA type II in 1,25-(OH)2D3 (10 nM)-treated cells compared to the control cells. Western blot analysis of 1,25-(OH)2D3-treated cells revealed a 4-fold increase in the cytosolic amount of the PKA regulatory subunit RII beta, whereas no changes were detected in the regulatory subunits RI alpha and RII alpha or the catalytic (C) subunit. Northern blot analyses showed a similar increase in RII beta mRNA in cells treated for 12 h with 1,25-(OH)2D3 (10 nM), and RII beta mRNA increased further to 10-fold above control cell level after 96 h of incubation. Iodide uptake was synergistically stimulated with both PKAI- and PKAII-directed pairs of cAMP analogs. The PKAI synergism was, however, inhibited by 1,25-(OH)2D3 treatment of the cells, whereas the PKAII synergism was unaffected. In conclusion, 1,25-(OH)2D3 attenuates both PKAI formation and PKAI-stimulated iodide uptake in rat thyroid FRTL-5 cells by increasing the level of RII beta without altering the other PKA subunit levels.