The effect of insulin in vitro on ATPase activities of nerve ending membranes and whole homogenates of rat cerebral cortex was studied. Membranes Na(+), K(+)-ATPase activity was not modified by 6.1 or 7.6 mU/ml insulin and it was inhibited 24-40% by 12.2-205 mU/ml insulin. Membranes Mg(2+)-ATPase activity was not modified by any of these insulin concentrations. In order to observe Na(+), K(+)-ATPase inhibition, prior to the enzyme assay, a preincubation period of the enzyme with insulin was required. The effect of insulin was dependent on the length of the enzyme incubation period. When the incubation period lasted 10 or 15 min no changes in ATPase activities were found; when the incubation period lasted 30 and 60 min, total and Na(+), K(+)-ATPase activities were inhibited 35-50%; Mg(2+)-ATPase activity appeared inhibited only after a 60 min incubation period. The addition of 205 mU/ml insulin inhibited by 40% Na(+), K(+)-ATPase activity of water whole homogenates but stimulated the enzyme up to 40% in sucrose whole homogenates. Both effects were observed in recently prepared cerebral cortex homogenates but not if the homogenates had been standing in the cold for 60 min before the enzyme assay. It is suggested that the insulin effect-stimulatory or inhibitory-is dependent on the integrity of some membranes of the cell.