Previous studies from this laboratory have established that addition of acetylcholine (ACh) or norepinephrine to 32P-labeled rabbit iris smooth muscle increases significantly the breakdown of triphosphoinositide (TPI) and that these stimulatory effects are blocked by atropine and phentolamine, respectively. The present studies were undertaken in order to show the effect of Ca++ on the ACh-stimulated breakdown of TPI ("TPI effect") in this tissue. Paired iris smooth muscles were prelabeled with 32Pi for 30 minutes at 37 degrees C in Ca++-free iso-osmotic salt medium. The prelabeled irises were then washed and incubated for 10 minutes in nonradioactive Ca++-free medium which contained 10 mM 2-deoxyglucose under various conditions. The phospholipids were isolated by means of two-dimensional thin-layer chromatography and their radioactivities were determined. In the absence of Ca++, 50 micrometer ACh increased TPI breakdown and phosphatidic acid (PA) labeling by 16 and 38%, respectively. In the absence of ACh, 0.75 micrometer Ca++ increased TPI breakdown and PA labeling by 11 and 20%, respectively. When both ACh and Ca++ were added, the increase in TPI breakdown and PA labeling rose to 32 and 74%, respectively. The labeling of phosphatidylinositol was found to be insensitive to the presence of Ca++. Ca++ was determined in the iris smooth muscle and it was found to contain 3.13 mumol of Ca++ per g of tissue. This was reduced by 80% after the muscle was washed and incubated in a medium which contained 0.25 micrometer ethyleneglycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). The TPI effect was abolished by 0.25 micrometer EGTA and restored when excess Ca++ (1.25 micrometer) was added. Concentrations of Ca++ as low as 50 micrometer provoked a TPI effect. Sr++ (2 micrometer), but not Ba++ or Mn++, was found to substitute partially for Ca++. Ionophore A-23187 (20 micrometer) was found to increase the breakdown of TPI and labeling of PA by 11 and 24%, respectively. High concentrations of Ca++ (20 mM) exerted similar effects. The increase in TPI breakdown and PA labeling in response to these agents, in contrast to the TPI effect in response to ACh, was not blocked by atropine. This suggests that the observed effects are not caused by the release of endogenous ACh from the muscle. A possible interpretation for the above observations on the role of Ca++ in the TPI effect at the postsynaptic membrane of the iris smooth muscle could be: (formula: see text).