Insulin-like growth factor I (IGF-I) plays a central role in skeletal growth by promoting bone cell replication and differentiation. Prostaglandin E2 (PGE2) and parathyroid hormone enhance cAMP production in cultured rat osteoblasts and stimulate IGF-I expression through a transcriptional mechanism mediated by cAMP-dependent protein kinase (PKA). We previously showed that PGE2 activated the transcription factor CCAAT/enhancer-binding protein delta (C/EBPdelta) in osteoblasts and induced its binding to a DNA element within the IGF-I promoter. We report here that a PKA-dependent pathway stimulates nuclear translocation of C/EBPdelta. Under basal conditions, C/EBPdelta was cytoplasmic but rapidly accumulated in the nucleus after PGE2 treatment (t(1/2) < 30 min). Nuclear translocation occurred without concurrent protein synthesis and was maintained in the presence of hormone. Nuclear localization required PKA and was blocked by a dominant-interfering regulatory subunit of the enzyme, even though C/EBPdelta was not a PKA substrate. Upon removal of hormonal stimulus, C/EBPdelta quickly exited the nucleus (t(1/2) < 12 min) through a pathway blocked by leptomycin B. Mutagenesis studies indicated that the basic domain of C/EBPdelta was necessary for nuclear localization and that the leucine zipper region permitted full nuclear accumulation. We thus define a pathway for PKA-mediated activation of C/EBPdelta through its regulated nuclear import.