We investigated the effect of Ca2+ overload on the phospholipase C-catalyzed hydrolysis of phosphoinositides in the rat left ventricular papillary muscle. Ca2+ overload on the papillary muscle was induced by treatment with 0.3 mM ouabain in Ca2+-containing medium following either Ca2+-containing or Ca2+-free superfusion. The phosphoinositide breakdown was evaluated by determining accumulations of [3H]inositol phosphates ([3H]IPs) in the tissues prelabeled with [3H]inositol. Ca2+ repletion following Ca2+-free superfusion resulted in a rapid but small increase in resting tension that was not followed by contracture, nor was it associated with a significant increase in [3H]IPs accumulations. Treatment with ouabain following Ca2+-containing superfusion increased resting tension after a lag period of several minutes and produced contracture associated with an increase in [3H]IPs accumulations. The ouabain induced increases in resting tension, and accumulations of [3H]IPs were significantly potentiated by prior Ca2+-free superfusion instead of Ca2+-containing superfusion. There was a significant positive correlation between increases in resting tension and the phosphoinositide breakdown. The increased resting tension and the accumulations of [3H]IPs were not antagonized by treatments with prazosin plus atropine or indomethacin, but were abolished by superfusion with Ca2+-free buffer solution. Although the enhanced phospholipase C-catalyzed hydrolysis of phosphoinositides appears to be a consequence rather than a cause of increased intracellular Ca2+, such a biochemical change may provoke a positive feedback mechanism to develop the muscle contracture through the putative intracellular messenger action of inositol triphosphate and diacylglycerol.