Inhibition of rat liver lysosomal phospholipases is one of the main events that leads to accumulation of tissue phospholipids during drug-induced phospholipidosis. Drug inhibition of lysosomal phospholipase A may occur by direct effects of drugs on the enzyme (or substrate) or by drug-induced increases in intralysosomal pH. Although beta-adrenoceptor blockers have not been reported to cause lipid storage, they do inhibit lysosomal phospholipase A. To investigate the structural requirements for drug inhibition, we studied the effects of six beta-adrenoceptor blockers on purified rat liver lysosomal phospholipase A1. The agents studied include: propranolol, timolol, metoprolol, practolol, atenolol and the combined alpha and beta adrenoceptor blocking agent, labetalol. The drugs varied by two logs in their abilities to inhibit phospholipase A1 activity. The relative inhibitory potencies were propranolol greater than labetalol much greater than timolol greater than metoprolol much greater than practolol greater than atenolol. Our studies identify drug hydrophobicity as a key determinant for phospholipase A1 inhibition. A strong negative correlation was noted between the octanol/water partition coefficients and IC50 for phospholipase inhibition (r = -0.91). The ability of propranolol to inhibit phospholipase A1 was identical for the d, l and the d and l stereoisomers.