Our previous studies have shown that cerebral microvessel endothelial cells (CMEC) express a Na-K-Cl cotransporter and that exposure of CMEC to astroglial cells causes a nearly 2-fold increase in activity of the cotransporter but only 1.5-fold increase in expression of cotransport protein [D. Sun, C. Lytle, and M. E. O'Donnell. Am. J. Physiol. 269 (Cell Physiol. 38): C1506-C1512, 1995]. This finding suggests that the astroglial cell effects may be mediated by mechanisms involving cotransporter activation in addition to increased protein expression. In the present study, we evaluated the role of protein phosphorylation in elevation of CMEC cotransport activity by astroglial cells and extracellular hypertonicity. We also examined the effects of protein phosphatase and protein kinase inhibitors on both cotransporter activity and phosphorylation in CMEC. The phosphorylation level of Na-K-Cl cotransport protein was quantitatively evaluated by immunoprecipitation analysis with the use of a monoclonal antibody to the cotransporter after 32P labeling of cultured CMEC. Activity of the cotransporter was assessed as bumetanide-sensitive K influx. We found that the phosphatase inhibitors calyculin A and okadaic acid significantly increased both cotransport activity and phosphorylation of cotransport protein. Activity and phosphorylation level of the cotransporter were also markedly increased by exposing the cells to astroglial cell-conditioned or hypertonic medium. Moreover, the astroglial-induced stimulation of the CMEC cotransporter was inhibited by the protein kinase inhibitor K-252a. These findings suggest that phosphorylation of cotransport protein plays an important role in regulation of Na-K-Cl cotransport activity and that astroglial-induced elevation of cotransport activity involves both phosphorylation-associated stimulation of cotransport activity and increased expression of the cotransporter protein.