The present study was performed to clarify second messenger signaling in parathyroid hormone (PTH)‐induced c‐fos gene expression, to characterize the participation of the c‐fos gene in the regulation of osteoblast proliferation and function as well as osteoclast‐like cell formation by PTH and to compare these effects of PTH with those of PTH‐related peptide (PTHrP). Both human (h) PTH‐(1‐34) and hPTHrP‐(1‐34) at 10^‐8 M induced a transient c‐fos gene expression to a similar degree in osteoblastic osteosarcoma cells, UMR‐106. N⁶, O²' ‐dibutyryl adenosine 3′, 5′‐cyclic monophosphate (dbcAMP) as well as Sp‐diastereoisomer of adenosine cyclic 3′,5′‐phosphorothioate (Sp‐cAMPS), an activator of cAMP‐dependent protein kinase (PKA), induced a weak c‐fos gene expression. Although Rp‐diastereoisomer of adenosine cyclic 3′,5′‐phosphorothioate (Rp‐cAMPS), an inhibitor of PKA, almost completely antagonized dbcAMP‐and Sp‐cAMPS‐induced expression of c‐fos gene, it did not cause an obvious inhibition of PTH‐or PTHrP‐induced expression. Phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C (PKC), induced an intense expression of the c‐fos gene, while 4α‐phorbol 12, 13‐didecanoate (4αPDD), incapable of activating PKC, and calcium ionophores (A23187 and ionomycin) did not. Protein kinase C inhibitor (H‐7, 50 μM) completely blocked the expression of the c‐fos gene by PTH as well as by PTHrP. Antisense oligodeoxynucleotides (as‐ODN) complementary to c‐fos mRNA, which have been shown to inhibit its mRNA translation, at 1 μM significantly antagonized PTH‐and PTHrP‐induced inhibition of [³H] thymidine incorporation and stimulation of osteoclast‐like cell formation in the presence of osteoblasts, but not an increase in alkaline phosphatase activity, compared to control oligodeoxynucleotides with same nucleotides as as‐ODN but with a random sequence. The present study indicates the involvement of PKC system in c‐fos gene expression by PTH as well as PTHrP and also indicates the involvement of the c‐fos gene in the regulation of bone cell physiology by PTH and PTHrP. © 1994 Wiley‐Liss, Inc.