We have previously shown that PTH induction of c-fos expression in the rat osteoblastic cell line UMR 106-01 requires the phosphorylation of cAMP response element-binding protein (CREB) at serine 133. Here we show that this event is not sufficient for induced transcriptional activity in UMR cells. Serine 129, but not the casein kinase II sites (serines 108, 111, 114, 117, and 121), also plays a role in the activation of CREB. First, by metabolically labeling an epitope-tagged CREB, we determined that, in addition to serine 133, other residues are phosphorylated in vivo. Using mutational analysis of a GAL4-CREB reporter system we demonstrate that serines 129 and 133 are both required for PTH-induced transcriptional activity, whereas the casein kinase II sites are not. Furthermore, PTH failed to induce transcriptional activity of GAL4-CREB in cells treated with genistein, a general tyrosine kinase inhibitor known to inhibit glycogen synthase kinase-3 (GSK-3) activity, or LiCl, the most specific GSK-3-inhibiting agent known, strongly implicating GSK-3beta in this process. Importantly, although genistein and LiCl each inhibit GSK-3beta activity, neither prevented the phosphorylation of serine 133 induced by PTH. Lastly, when serine 129 is replaced with a negatively charged aspartic acid, LiCl has no effect on the PTH-induced trans-activation of CREB. We propose that GSK-3beta phosphorylates CREB at serine 129 and thus is required for the increased transcriptional activity of CREB in response to PTH.