The effects of insulin on monovalent cation transport and on Na-K-ATPase activity from intact cells, tissue homogenates, and purified enzyme of the avian salt gland were studied. Monovalent cation active transport, measured by ouabain-inhibitable 86Rb+ uptake, was significantly increased (21.9 +/- 7.3% SE) in tissue slices exposed to insulin (100 mU/ml) for 15 min. A small but significant (12.2 +/- 1.9%) increase in Na-K-ATPase activity was similarly observed after salt gland tissue slices were exposed to insulin. This increase in enzymatic activity did not occur when broken-cell homogenates were exposed to insulin. Purified preparations of Na-K-ATPase showed no insulin enhancement of activity either in the presence of optimal or less than fully activating Na+ and ATP concentrations. Na-K-ATPase activity was the same in detergent-activated homogenates of both control and insulin-treated slices, consistent with insulin activation of existing enzyme sites. These data support the hypothesis that at least part of the increase in monovalent cation active transport produced by insulin is related to enhanced Na-K-ATPase activity and indicate that the latter phenomenon is dependent on some components or properties of the intact cell.