Recent work has established that various bone-resorbing hormones are able to activate phosphoinositide metabolism as well as eicosanoid production in osteoblast-like cells, although the relationship between these pathways is unclear. We used pertussis toxin and indomethacin to inhibit the stimulation of [3H]arachidonic acid release and [3H]phosphoinositide turnover caused by treating primary cultures of mouse osteoblasts with fetal calf serum. We found (1) that pertussis toxin and indomethacin each inhibited both pathways and (2) that although pertussis toxin inhibited [3H]arachidonic acid release to a greater extent than indomethacin, [3H]inositol phosphate accumulation was inhibited rather more effectively by indomethacin. These data suggest that whereas ligands in fetal calf serum activate [3H]arachidonic acid release largely directly via the action of a pertussis-sensitive G protein, activation of phosphoinositidase C is indirect, being substantially dependent upon eicosanoid production. These experiments suggest that serial activation of phospholipase A2 and phosphoinositidase C may occur in osteoblasts and that only the former enzyme is regulated by a pertussis toxin-sensitive G protein.