The current study sought to define the molecular mechanisms involved in the cross talk between 1,25dihydroxyvitamin D₃ (1,25(OH)₂D₃) and activators of protein kinase C (PKC) in the regulation of 25(OH)D₃ 24-hydroxlyase (24(OH)ase). Transfection of the h24(OH)ase promoter construct [-5,500/-22 luciferase; vitamin D responsive elements at (-294/-274 and -174/-151); AP-1 site at -1167/-1160] in VDR transfected COS-7 cells resulted in strong activation by 1,25(OH)₂D₃. In these cells, co-treatment with the protein kinase C (PKC) activator, 12-o-tetradecanoylphorbol-13-acetate (TPA) and 1,25(OH)₂D₃ yielded a 27 fold increase in luciferase activity which was 2-3 fold greater than activation obtained with 1,25(OH)₂D₃ alone (p<0.05).Similar results were observed using LLCPK-1 kidney cells, suggesting that the previously observed enhancement of 1,25(OH)₂D₃ induced renal 24(OH)ase mRNA and activity by PKC activation occurs at the level of transcription. The functional cooperation between PKC activation and VDR was not found to be mediated by the AP-1 site in the h24(OH)ase promoter nor by enhanced binding of GRIP or DRIP205 to VDR and was also not due to PKC mediated phosphorylation of VDR on serine 51. Our study demonstrates that in LLCPK-1 kidney cells the PKC enhancement of 1,25(OH)₂D₃ stimulated transcription may be due, in part, to an increase in VDR concentration. In addition, inhibitors of the mitogen activated protein kinase (MAPK) pathway were found to decrease the TPA enhancement (p<0.05). Since activation of MAPK has been reported to result in the phosphorylation of SRC-1 and in functional cooperation between SRC-1 and CREB binding protein, we propose that the potentiation of VDR mediated transcription may also be mediated through changes in the phosphorylation of specific VDR coregulators.