Whole brain spheroids provide a suitable model to study neurodevelopment. In the literature a role for the nitric oxide (NO)-cyclic guanosine 3',5'-monophosphate (cGMP) signalling pathway during development has frequently been suggested. In this study we investigated whether functional cGMP pathways were present in differentiated spheroids. In 3-week-old spheroids soluble guanylate cyclase was stimulated with N-methyl D-aspartic acid or sodium nitroprusside (NO donor). The results showed that the NO synthase-cGMP pathway is present in the culture system. Soluble guanylate cyclase-dependent cGMP formation was found in NO synthase containing neurons, in neurons of the GABAergic, glutamatergic and cholinergic system, and in astroglia and oligodendroglia. Activation of particulate guanylate cyclase by atrial natriuretic peptide also triggered an increase in cGMP production. Particulate guanylate cyclase was found in astroglia and in microglia as well as in glutamic acid decarboxylase and calbindin containing structures and neuronal NO synthase containing neurons. Chronic inhibition of NO synthase during culture development had no effect on soluble or particulate guanylate cyclase functioning. Similarly, inhibition of soluble guanylate cyclase during culture development did not have any effect on NO synthase and particulate guanylate cyclase functioning. It is concluded that NO synthase and both soluble and particulate guanylate cyclase are present in whole brain spheroid cultures and that their activity can be influenced by several stimuli. The spheroid culture system constitutes a suitable model to study the NO-cGMP pathway during brain development in mammals.