The effects of ATP, U-73122, apyrase, and saline shear stress on [Ca2+]i homeostasis were studied in fura-2 loaded, mouse fibroblast cells (L929), both in suspension and plated on glass. Release of internal Ca2+ was induced by ATP, via a receptor identified pharmacologically as a P2U type. In single cells, low concentrations of ATP evoked [Ca2+]i oscillations. These events were blocked by the putative phospholipase C inhibitor, U-73122 (but not by the inactive analog U-73343) and by the ATP/ADPase, apyrase. In addition, both these agents reduced the [Ca2+]i of unstimulated cells, especially after stirring, and blocked spontaneously occurring [Ca2+]i oscillations, which suggested an already activated state of the ATP receptor, independent from exogenous stimulations. Moreover, it was found that stirring of the cells was correlated with a steady accumulation of inositol phosphates, also blockable by apyrase, and that [Ca2+]i mobilization could be induced by puffs of saline in single cells. The transition to a Ca(2+)-free environment also provoked [Ca2+]i oscillations, most likely via the increase in ATP4- concentration. This evidence suggests that endogenous ATP is released from L fibroblasts in response to fluid shear stress, and this results in an autocrine, tonic up-regulation of the phosphoinositide signaling system and an ensuing alteration in Ca2+ homeostasis. Up until now, such a response to shear stress was believed to be unique to endothelial cells.