In earlier studies, the neural cell adhesion molecule, N-CAM, was found to inhibit the proliferation of rat astrocytes both in vitro and in vivo. To identify the gene targets involved, we used subtractive hybridization to examine changes in gene expression that occur after astrocytes are exposed to N-CAM in vitro. While the mRNA levels for N-CAM decreased after such treatment, the levels of mRNAs for glutamine synthetase and calreticulin increased. Since glutamine synthetase and calreticulin are known to be involved in glucocorticoid receptor pathways, we tested a number of steroids for their effects on astrocyte proliferation. Dexamethasone, corticosterone, and aldosterone were all found to inhibit rat cortical astrocyte proliferation in culture in a dose-dependent manner. RU-486, a potent glucocorticoid antagonist, reversed the inhibitory effects of dexamethasone. These observations prompted the hypothesis that the inhibition of proliferation by N-CAM might be mediated through the glucocorticoid receptor pathway. Consistent with this hypothesis, the inhibition of astrocyte proliferation by N-CAM was reversed in part by a number of glucocorticoid antagonists, including RU-486, dehydroepiandrosterone, and progesterone. In transfection experiments with cultured astrocytes, N-CAM treatment increased the expression of a luciferase reporter gene under the control of a minimal promoter linked to a glucocorticoid response element. The enhanced activity of this construct stimulated by N-CAM was abolished in the presence of RU-486. The combined data suggest that astrocyte proliferation is in part regulated by alterations in glucocorticoid receptor pathways.