We have previously reported that insulin/insulin-like growth factor (IGF)-I induced the alpha 1 isoform of Na+,K(+)-ATPase in cultured astrocytes. In this study the effects of insulin/IGF-I on Na+,K(+)-ATPase activity and cell proliferation were examined in astrocytes cultured under the various conditions, to test the possible involvement of the enzyme activity in the mitogenic action of IGF-I on astrocytes. Insulin increased Na+,K(+)-ATPase activity and stimulated cell proliferation in subconfluent astrocytes (cultured for 7-14 days in vitro). In contrast, these effects were not observed in confluent cells (cultured for 28 days). Furthermore, insulin stimulated neither the enzyme activity nor [3H]thymidine incorporation in astrocytes preincubated in fetal calf serum-free medium for 2 days (quiescent cells) and treated with dibutyryl cyclic AMP (differentiated cells). The increases in Na+,K(+)-ATPase activity and expression of the alpha 1 mRNA preceded the mitogenic effect. 125I-IGF-I binding experiment showed that all the cells used here had similar binding characteristics. The insulin-induced increase in enzyme activity was not affected by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and it was observed even in Ca(2+)-free medium. The stimulation by IGF-I of [3H]thymidine incorporation was attenuated by ouabain and a low external K+ level. These findings suggest that stimulation of Na+,K(+)-ATPase activity is involved in the mitogenic action of IGF-I on cultured astrocytes.