The membrane perturbational actions of pentobarbital, methohexital, and phenobarbital were analyzed in terms of their effects on the incorporation of group-specific chemical probes into protein and phospholipid structural components and on the activity of various functionally distinct ATPases in erythrocyte and brain synaptosomal membranes. When compared at concentrations producing similar degrees of antihemolysis in red cells, the three analogues differed most markedly in the nature and degree of the structural and functional alterations induced in membrane proteins, with the most highly lipophilic agent, methohexital, being frequently, although not invariably, the most potent. Comparison of the effects of pentobarbital on characteristics of trinitrobenzenesulfonic acid and 5,5′-dithiobis-(2-nitrobenzoic acid) incorporation into erythrocyte membranes with results previously obtained using other anaesthetics at concentrations producing 50% antihemolysis showed that pentobarbital behaves similarly to neutral general anaesthetics but differently from cationic local anaesthetics. Our findings suggest that the membrane perturbational characteristics of barbiturates may contribute to their diverse and complex actions on excitable tissues.