We tested the effects that pertussis toxin had on bone resorption mediated by cAMP-dependent and cAMP-independent stimuli in 19-day-old fetal rat long bones. Agents that stimulate cAMP were PTH, prostaglandin E2, and calcitonin. Agents that act independent of cAMP were: phorbol 13-myristate 12-acetate (PMA), 1,25-dihydroxyvitamin D3, murine interleukin-1 alpha, osteoclast-activating factor, and human tumor necrosis factor-alpha. Pertussis (1-10 ng/ml) produced a dose-related inhibition of resorption in unstimulated control cultures. The inhibitory effect was not associated with changes in either [3H]thymidine or [3H]proline incorporation into bones. beta-Glucuronidase activity in the medium was decreased. PMA was the only agonist whose resorptive effect was completely blocked by pertussis. The resorptive response to other stimulators was reduced, but treated/control ratios usually remained the same or increased because of the greater effect of pertussis on control resorption. There was a partial inhibition of the resorptive effect of low doses of prostaglandin E2 (10 nM), but increasing the concentration of agonist overcame the inhibition. Pertussis did not enhance the sensitivity of bones to calcitonin. Pertussis enhanced the cAMP response to PTH, but had no effect on basal cAMP production. Since PMA was inhibited by pertussis while agents that may act through cAMP-mediated or phosphatidylinositol pathways were not affected, we hypothesize that a protein kinase-C dependent pathway can modulate bone resorption.