Radiographic contrast media (RCM) used in the subarachnoid space are associated with occasional adverse reactions. This study examines the possibility that RCM reactions are caused by interactions with the plasma membrane phosphatidylinositol (PI) second messenger system. Isolated nerve endings, known as synaptosomes, were produced from rat brain homogenates. The synaptosomes were then incubated with RCM to determine if 32Pi labeling of the PIs or the uptake of 45Ca were influenced in a manner consistent with known mechanisms. The RCM metrizamide, iopamidol, iodixanol, and iotrol (but not iohexol) increased the 32Pi labeling. Hyperosmolality produced large increases in phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4, 5,-bisphosphate (PIP2) labeling. In the non-depolarized state iodixanol, but not metrizamide or iohexol, caused a time-dependent increase in 45Ca uptake. Iodixanol, iohexol, and metrizamide also augmented the veratrine-stimulated uptake of calcium, but none of the RCM affected the uptake of Ca resulting from potassium depolarization. The increased 32Pi labeling of the PIs caused by RCM is not directly related to Ca uptake, because the direction of change is wrong. RCM perturbations of the plasma membrane may cause an inhibition of other membrane components and systems. Hyperosmolality also may cause inhibition of membrane components. It is not known if these effects are important in clinically observed RCM toxicity.