Agrin is a heparan sulfate proteoglycan of the extracellular matrix and is known for organizing the postsynaptic differentiation of the neuromuscular junction. Increasing evidence also suggests roles for agrin in the developing CNS, including the formation and maintenance of the blood-brain barrier. Here we describe effects of agrin on the expression and distribution of the water channel protein aquaporin-4 (AQP4) and on the swelling capacity of cultured astrocytes of newborn mice. If astrocytes were cultured on a substrate containing poly DL-ornithine, anti-AQP4 immunoreactivity was evenly and diffusely distributed. If, however, astrocytes were cultured in the presence of agrin-conditioned medium, we observed an increase in the intensity of AQP4-specific membrane-associated staining. Freeze-fracture studies revealed a clustering of orthogonal arrays of particles, representing a structural equivalent of AQP4, when exogenous agrin was present in the astrocyte cultures. Neuronal and non-neuronal agrin isoforms (agrin A0B0 and agrin A4B8, respectively) were able to induce membrane-associated AQP4 staining. Water transport capacity as well as the density of orthogonal arrays of intramembranous particles was increased in astrocytes cultured with the neuronal agrin isoform A4B8, but not with the endothelial and meningeal isoform A0B0. RT-PCR demonstrated that agrin A4B8 increased the level of the M23 splice variant of AQP4 and decreased the level of the M1 splice variant of AQP4. Implications for the regulation and maintenance of the blood-brain barrier including oedema formation under pathological conditions are discussed.