Murine MC3T3-E1 and MC-4 cells were stably transfected with -371/+70 bp of the murine cyclooxygenase-2 (COX-2) promoter fused to a luciferase reporter (Pluc371) or with Pluc371 carrying site-directed mutations. Mutations were made in (1) the cAMP response element (CRE) at -57/-52 bp, (2) the activating protein-1 (AP-1)-binding site at -69/-63 bp, (3) the nuclear factor of activated T-cells (NFAT)-binding site at -77/-73 bp, and (4) both the AP-1 and NFAT sites, which comprise a composite consensus sequence for NFAT/AP-1. Single mutation of CRE, AP-1, or NFAT sites decreased parathyroid hormone (PTH)-stimulated COX-2 promoter activity 40% to 60%, whereas joint mutation of NFAT and AP-1 abrogated the induction. On electrophoretic mobility shift analysis, PTH stimulated binding of phosphorylated CREB to an oligonucleotide spanning the CRE and binding of NFATc1, c-Fos, and c-Jun to an oligonucleotide spanning the NFAT/AP-1 composite site. Mutation of the NFAT site was less effective than mutation of the AP-1 site in competing binding to the composite element, suggesting that cooperative interactions of NFATc1 and AP-1 are more dependent on NFAT than on AP-1. Both PTH and forskolin, an activator of adenylyl cyclase, stimulated NFATc1 nuclear translocation. PTH- and forskolin-stimulated COX-2 promoter activity was inhibited 56% to 80% by calcium chelation or calcineurin inhibitors and 60% to 98% by protein kinase A (PKA) inhibitors. These results indicate an important role for the calcium-calcineurin-NFAT signaling pathway in the PTH induction of COX-2 and suggest that cross-talk between the cAMP/PKA pathway and the calcium-calcineurin-NFAT pathway may play a role in other functions of PTH in osteoblasts.