The effects of OST-766, an inhibitor of vacuolar H+-ATPase activity, on adenylyl cyclase and phospholipase C activity were explored in the osteoblast cell line ROS 17/2.8. In fresh homogenates of ROS 17/2.8 cells, OST-766 inhibited adenylyl cyclase activity (ACA) in response to guanine nucleotide and forskolin but had no effect on basal ACA. OST-766 enhanced the basal generation of IP2, but not that formed in response to Ca2+ or guanine nucleotides. In marked contrast, incubation of intact ROS 17/2.8 cells with OST-766 for at least 48 hours resulted in an increase in basal ACA as well as in response to PTH, guanine nucleotides and forskolin. Under similar conditions, the compound also increased IP1, IP2 and IP3 generation in response to guanine nucleotides and Ca2+. Levels of the guanine nucleotide binding proteins Gs and Gi were also increased in OST-766-treated cells. The results suggest that the actions of this H+-ATPase inhibitor include effects on osteoblasts through PTH-sensitive signal transduction pathways.