1,25-Dihydroxyvitamin D3 (1,25D3) inhibits cell growth and induces differentiation in many cell systems by inhibition of c-myc gene expression. In the human medullary thyroid carcinoma cell line (TT), c-myc gene expression appears to be closely related to cell proliferation and differentiation. TT cells are also a well known target system for 1,25D3, which inhibits calcitonin (CT) gene expression in these cells. So far, no direct cis-acting vitamin D-responsive element could be identified in the promoter region of the CT gene. We, therefore, investigated potential indirect mechanisms of 1,25D3-mediated CT gene expression by examining the hormone's effects on proliferation. In contrast to its well established antiproliferative action in other cell systems, addition of 1,25D3 to TT cells led to a 2.3-fold stimulation of DNA synthesis, which was maximal after 48 h and was preceded by a 4.8-fold increase in c-myc gene expression. c-Myc antisense DNA oligomers abolished the proliferative effect of 1,25D3, but not the latter's inhibition of CT gene expression. Here we present evidence that activation of c-myc gene expression mediates 1,25D3-stimulated TT cell proliferation, but not the 1,25D3-induced inhibition of CT gene expression.