Water balance between cells and extracellular compartments is essential for proper functioning of the central nervous system, as demonstrated by its perturbations in pathological conditions. Aquaporin 4 (AQP4) is the predominant water channel in brain and spinal cord, where it is present mainly on astrocytic endfeet contacting vessels. A role in water homeostasis control has been proposed also for the extracellular matrix, that in brain consists mainly of chondroitin sulfate proteoglycans (CSPGs). Using cytochemical and immunocytochemical techniques, we investigated their distribution in rodent spinal cord, to better understand the role of these two classes of molecules. The results show that in spinal gray matter AQP4 labeling is intense in all perivascular profiles and (1) displays a marked dorsoventral gradient in the neuropil; and (2) coexists extensively with glial glutamate transporter-1 (GLT-1) but scarcely with glial fibrillary acidic protein (GFAP). In white matter the overlap between AQP4, GLT-1, and GFAP is almost complete. Ultrastructural examination shows that AQP4-labeled astrocytic processes surround blood vessels, neuronal perikarya and processes, and both asymmetric and symmetric synapses, indicating that the protein may be involved in the regulation of water fluxes around both inhibitory and excitatory synapses. CSPGs, visualized by labeling with Wisteria floribunda agglutinin, show a distribution complementary to that of AQP4, being absent or weekly expressed in AQP4-enriched areas. These findings suggest that different mechanisms may contribute to the regulation of water homeostasis in different spinal cord regions. © 2005 Wiley-Liss, Inc.