The inhibition of canine renal sodium potassium adenosine triphosphatase (ATPase) by neomycin was examined. Neomycin inhibited ATPase nearly maximally at 0.02 mM. The inhibition was temperature dependent with a decrease in inhibition occurring at temperatures below 21 degrees C, a temperature which corresponded to a change in activation energy of the ATPase as determined by Arrhenius plot. Preincubation of the ATPase with phosphoinositides was found to prevent the inhibition by neomycin. Other phospholipids were not found to prevent the inhibition. These results indicate a possible interaction between neomycin and the phosphoinositides of the ATPase complex.