Mechanical stimulation of one mammary tumor cell in culture induced an increase in its intracellular calcium concentration which spread to surrounding cells. The increase in calcium can also be induced by addition of a solution in which cultured mammary tumor cells were stimulated by repeated pipetting (solution after pipetting cells, SAPC). The activity of the SAPC was completely abolished by treatment with snake venom phosphodiesterase or pyrophosphatase. Uridine triphosphate (UTP), uridine diphosphate (UDP) and ATP (1 mu M each) were detected in the SAPC, whereas 5'-UMP and 5'-AMP were produced by phosphodiesterase digestion. A mixture of UTP, UDP and ATP (1 mu M each) elicited a calcium response which was comparable to that induced by SAPC, while UTP, UDP or ATP alone at 1 mu M elicited a small increase in calcium concentration in mammary tumor cells. Suramin, a competitive antagonist of P-2 purinoceptors, diminished the spreading of the calcium wave induced by mechanical stimulation, It also blocked the responses to SAPC, UTP, UDP and ATP. These findings suggest that the mechanical stimulation results in the release of UTP, UDP and ATP into the extracellular space which mediates induction of the spreading calcium response via P-2U-type purinoceptors.