The osteocalcin gene encodes an osteoblast-specific protein that is induced with the onset of mineralization at late stages of differentiation. Several transcriptional regulators have been characterized that control the transcription of osteocalcin, including activator protein 1 (AP-1) family members such as the Fra2/JunD heterodimer. We have previously shown that the c-Jun homodimer activates transcription from the murine osteocalcin proximal promoter and that this response is potentiated by the alpha chain of the nascent polypeptide-associated complex (alphaNAC) transcriptional coactivator. We now further explore the mechanisms involved and show that c-Jun binds two cryptic AP-1 sites within the proximal promoter of osteocalcin and that this binding is strictly alphaNAC-dependent. Chromatin immunoprecipitation (ChIP) confirmed that c-Jun occupies its binding sites within the osteocalcin 5'-flanking region in living osteoblasts. Interestingly, the ChIP assay revealed that both JunB and JunD also bind the osteocalcin promoter. JunD, but not JunB, stimulated osteocalcin gene transcription in transient transfection assays, but this effect was not potentiated by alphaNAC. Thus, the c-Jun and JunD family members utilize distinct mechanisms that implicate differential interaction with transcriptional coactivators to regulate osteocalcin expression.