Nitric oxide plays an important role in many biological processes including the cardiovascular, immune, reproductive and nervous systems. There is much interest surrounding this molecule and the enzyme responsible for its synthesis, nitric oxide synthase. In order to investigate the role of nitric oxide in various biological processes it is necessary to be able to identify the released molecule itself as well as nitric oxide synthase. Detection of nitric oxide synthase was achieved by optimisation of the immunohistochemical localisation of the neuronal isoform of the enzyme in primary cultures of rat ventral mesencephala and in fixed adult rat brain sections. Furthermore, using 4,5-diaminofluorescein diacetate, we have directly visualised endogenously produced nitric oxide in mesencephalic cultures and demonstrated the potential use of this indicator for visualising nitric oxide produced in vivo.