The effect of the inositol phospholipid-binding antibiotic neomycin was studied on high-affinity GTPase in human platelet membranes. At low concentrations (up to 1 mM), neomycin by itself stimulated a high-affinity GTPase. This GTPase stimulation was additive with that caused by the hormonal factors, prostaglandin E1 and epinephrine, but not with thrombin. At concentrations higher than 1 mM, neomycin reduced control GTPase activity and eliminated the stimulation caused by thrombin. The data suggest that neomycin by a presently unknown mechanism can regulate activity states of signal transducing GTP-binding proteins.