Astrocytes are important in the development and maintenance of functions of the CNS, acting in cooperation with neurons and other glial cells. The glycans on astrocyte membrane are believed to play important roles in cell-cell communication. Plant lectins are useful probes, because the lectins can bind to certain cell surface receptors and elicit cellular responses that are normally activated by endogenous ligands for those receptors. In the present study, we investigated the effect of Datura stramonium agglutinin (DSA) on astrocytes and characterized several molecular events. The addition of DSA to a culture of flat, polygonal, immature astrocytes derived from the neonatal rat cerebellum caused the cells to become stellate in shape, similar to astrocytes observed in vivo, concomitant with an increase in expression of astrocyte-specific intermediate filament (glial fibrillary acidic protein [GFAP]) and inhibition of proliferation. These results indicate that DSA binds to astrocytes and triggers differentiation. We also found a decrease in the extent of tyrosine-phosphorylation of a 38-kDa protein. To elucidate the molecular events during astrocyte differentiation, we examined the effects of various signal transduction inhibitors on the transformation from the polygonal to stellate shape (stellation). Interestingly, only tyrosine phosphatase inhibitors, orthovanadate and phenylarsine oxide, showed an inhibitory effect. Our results suggest that DSA induced astrocyte differentiation acts via tyrosine dephosphorylation.