The gap junction protein connexin 43 (Cx43) mediates communication between osteoblasts and is important for maximal PTH responsiveness. We examined the role of the Cx43 promoter and messenger RNA 3' untranslated region (UTR) in conferring responsiveness to PTH in the rat osteosarcoma cell line UMR-106. PTH induced a 4-fold increase in luciferase activity of a reporter construct containing 1.6 kb 5' of the transcription start site. Deletion analysis of the promoter localized responsive sequences to between -31 to +1 bp. PTH treatment of transgenic mice containing the 1.6 kb promoter luciferase construct induced increases in luciferase and Cx43 immunoreactivity in bone cells underlying the tibial growth plate. The full Cx43 3'UTR conferred a 3-fold response to PTH when placed 3' of a CMV-luciferase construct. Deletion analysis localized responsive sequences to between 2510 and 3132 of the 3'UTR. Cloning of this segment 5' of the CMV promoter disrupted the PTH response, indicating this sequence does not function as an enhancer. Sequences within the promoter conferred responsiveness to forskolin, whereas the 3'UTR responded to both TPA and forskolin. These data indicate that PTH responsive sequences are present in the Cx43 promoter and 3'UTR, suggesting that transcriptional and posttranscriptional pathways operate to regulate PTH-induced Cx43 expression in osteoblast cells.