Parathyroid hormone (PTH) regulates osteoblastic differentiation and activity and exerts different overall skeletal effects in vivo depending upon the schedule and dose of administration. In clonal Wt9 murine osteoblastic cells, mRNA and protein levels of the CITED1 transcriptional coactivator were strongly up-regulated by hPTH(1–34). Stimulation of CITED1 mRNA by PTH was transient, peaking at 4 h, concentration-dependent, and blocked by actinomycin D but not cycloheximide. The stimulation was mimicked by forskolin, phorbol ester and the cAMP-selective PTH analog [G¹,R¹⁹]hPTH(1–28), and inhibited completely by the PKA inhibitor, H89 and partially by phorbol ester-induced PKC depletion. Increased CITED1 expression was not maintained during persistent (24-h) PTH exposure. Cultured primary calvarial osteoblasts from neonatal homozygous or hemizygous CITED1-knockout (KO) mice achieved 2-fold greater mineralized nodule formation in comparison to wild-type (WT) osteoblasts. This effect was blocked by restoration of CITED1 expression via adenoviral gene transfer. Intermittent administration of hPTH(1–34) (10 nM, for 4 h every 48 h) for 3–6 wk increased mineralization up to 2-fold over basal levels in both WT and CITED1 KO mouse calvarial-cell cultures. Whereas the cAMP-selective [G¹,R¹⁹]hPTH(1–28) analog (at 100 nM, equivalent to 10 nM hPTH(1–34)) did not simulate mineralization in WT cultures, it was twice as effective as hPTH(1–34) in CITED1 KO cultures. Thus, CITED1 negatively regulates osteoblastic differentiation in vitro and inhibits the cAMP-dependent stimulation of differentiation by intermittent PTH. We conclude also that PTH receptor signaling pathways independent of cAMP restrain osteoblastic differentiation, an effect normally obscured in the presence of CITED1 but revealed in its absence.