Osteocalcin (OC) is a bone-specific extracellular matrix protein expressed by mature osteoblasts during late stages of differentiation. Previous studies have shown that forskolin, an activator of adenylate cyclase, stimulated OC production. Because PTH has been shown to activate several intracellular signal transduction pathways including cAMP, inositol phosphate and intracellular calcium mobilization, we investigated whether PTH action on cAMP accumulation leads to OC promoter activation. The rat OC promoter (1095 bp) was cloned into the promoterless luciferase gene reporter vector. The transcriptional activity of the rat OC promoter was evaluated after transfection of SaOS-2, an osteosarcoma cell line, with the OC promoter followed by treatment with PTH. Maximal OC promoter activity was observed within 4–8 h after the addition of 10⁻⁸m PTH, whereas very little induction was seen after 24 and 48 h of treatment. The induction of OC promoter activity by PTH was concentration dependent. PTH analogs (PTH 1–84, PTH 1–34, and PTH 1–31) that stimulate intracellular cAMP accumulation, induced OC promoter activity, whereas other PTH analogs (PTH 3–34, PTH 7–34, PTH 13–34, and PTH 53–84) that do not stimulate cAMP production had no effect on OC promoter activation. Furthermore, PTH activation of the OC promoter was significantly enhanced in the presence of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Inactivation of cAMP-dependent protein kinase A activity by either a selective protein kinase A inhibitor, H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5 isoquinolinesulfonamide), or antisense oligonucleotide directed against the regulatory subunit of cAMP-dependent protein kinase A, led to a corresponding loss of OC promoter activation by PTH. 5′ deletion analysis of the OC promoter demonstrated that the promoter (1095 bp) exhibited the greatest response to PTH, whereas the −198 bp construct of the OC promoter, containing only one cAMP response element and OC box, was no longer responsive. The constructs with further deletions (−120, −92, and −74) retained PTH responsiveness, but to a lesser extent. In summary, our results indicate that PTH activation of the OC promoter is a rapid event and mediated by the cAMP-dependent protein kinase A pathway. Although the novel cAMP response region overlapping the OC box is required for activation, full activation may require several cis-acting cAMP response elements or other response elements.