To understand components shaping the neuronal environment we studied the astroglial cells in the zebrafish brain using immunocytochemistry for structural and junctional markers, electron microscopy including freeze fracturing, and probed for the water channel protein aquaporin-4. Glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) showed largely overlapping immunoreactivity: GFAP in the main glial processes and GS in main processes and smaller branches. Claudin-3 immunoreactivity was spread in astroglial cells along their major processes. The ventricular lining was immunoreactive for the tight-junction associated protein ZO-1, in the telencephalon located on the dorsal, lateral, and medial surface due to the everting morphogenesis. In the tectum, subpial glial endfeet were also positive for ZO-1. Correspondingly, electron microscopy revealed junctional complexes between subpial glial endfeet. However, in freeze-fracture analysis tight junctional strands were not found between astroglial membranes, either in the optic tectum or in the telencephalon. Occurrence of aquaporin-4, the major astrocytic water channel in mammals, was demonstrated by polymerase chain reaction (PCR) analysis and immunocytochemistry in tectum and telencephalon. Localization of aquaporin-4 was not polarized but distributed along the entire radial extent of the cell. Interestingly, their membranes were devoid of the orthogonal arrays of particles formed by aquaporin-4 in mammals. Finally, we investigated astroglial cells in proliferative areas. Brain lipid basic protein, a marker of early glial differentiation but not GS, were present in some proliferation zones, whereas cells lining the ventricle were positive for both markers. Thus, astroglial cells in the zebrafish differ in many aspects from mammalian astrocytes.