Aminoglycosides are able to inhibit phosphatidylinositol phospholipase C activity in a soluble fraction of rat renal tissue. The Km of the enzyme with respect to phosphatidylinositol is 0.53 mM and Vmax was 0.671 mumol of phosphatidylinositol cleaved per hr per mg of protein. The enzyme is calcium activated and inhibited by ethylene glycol bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid. Streptomycin, amikacin, kanamycin, tobramycin, gentamicin and neomycin inhibit enzyme activity in the same rank order as their ability to produce nephrotoxicity. The 50% inhibitory concentrations ranged from 0.03 mM for neomycin to 0.38 mM for streptomycin. Lineweaver Burk plots indicate competitive inhibition with a possible relation to the calcium activation. It is possible that aminoglycosides may inhibit phosphatidylinositol phospholipase C in vivo and that this inhibition may be related to the nephrotoxicity of aminoglycosides.