In the central nervous system nitric oxide appears to be critically involved in a number of physiological and pathological processes. Although there is convincing evidence for expression of nitric oxide synthase in cultured glial cells, demonstration of this enzyme in glial cells in situ remained largely unsatisfactory. In the present study we applied immunostaining to freeze-dried sections of snap-frozen hippocampi and cerebella of rats and to sections of freeze-dried brain tissue in order to minimize diffusion artefacts and thus to obtain more precise information about the true in situ localization of nitric oxide synthase. Here we show that astrocytes and Bergmann glia react strongly with antibodies raised against cerebellar nitric oxide synthase and against a type I nitric oxide synthase-specific C-terminal peptide, respectively. This finding was further substantiated by histochemical localization of NADPH-diaphorase activity in astrocytes and Bergmann glia as well as by immunoreactivity of both types of glia cells with antibodies to the NADPH-delivering enzyme glucose-6-phosphate dehydrogenase. We conclude, that astrocytes are important sites of nitric oxide synthase I in brain, suggesting that these cells might use nitric oxide as gaseous messenger molecule for various aspects of glia-neuron signalling. o 1996 Wiley-Liss, Inc.