Insulin-like growth factor I (IGF-I) is thought to stimulate bone resorption indirectly through a primary effect on osteoblasts, which in turn activate osteoclasts by as-yet-unidentified mechanisms. Small decreases in extracellular pH (pH_o) dramatically increase the resorptive activity of osteoclasts. Our purpose was to characterize the effect of IGF-I on acid production by osteoblastic cells. When confluent, UMR-106 osteoblast-like cells and rat calvarial cells acidified the compartment beneath them. Superfusion with IGF-I caused a further decrease in pH_o. To investigate the mechanism, we monitored acid efflux from subconfluent cultures. IGF-I rapidly increased net efflux of H⁺ equivalents in a concentration-dependent manner. IGF-II (10 nM) evoked a smaller response than IGF-I (10 nM). The response to IGF-I was partially dependent on extracellular Na⁺, but not glucose, and exhibited little if any desensitization. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, abolished the response to IGF-I but not to parathyroid hormone. Thus IGF-I enhances acid efflux from osteoblastic cells, via a signaling pathway dependent on activation of phosphatidylinositol 3-kinase. In vivo, acidification of the compartment between the osteogenic cell layer and the bone matrix may affect diverse processes, including mineralization and osteoclastic bone resorption.