Experiments were carried out in urethane-an-anesthetized rats to examine the effect of nitric oxide (NO) on neuronal activity within the dorsolateral sector of the midbrain periaqueductal grey matter (PAG), an area which is rich in NO-synthesizing neurones. NADPH-dependent diaphorase histochemistry revealed small NO synthase-containing perikarya, 15.4 +/- 3.1 microns (mean +/- SEM) in diameter, in a longitudinal column in the dorsolateral sector of the PAG. The labelled cell bodies were surrounded by a dense meshwork of stained fibres and processes in which unlabelled neurones were embedded. In order to establish whether NO was generated when NO donors were ejected iontophoretically from micropipettes, a chemiluminescence method was used to estimate the output of NO in vitro after iontophoresis of two chemically different classes of NO donor: the sydnonimine 3-morpholino-sydnonimin-hydrochloride (SIN 1) and the nitrosothiol S-nitroso glutathione (SNOG). Iontophoresis of both NO donors into 200 microliters aliquots of 165 mM NaCl using ejection currents between 6000 and 18,000 nA.min produced a current-related increase in the concentration of NO. Iontophoresis of SIN 1 in vivo produced a reproducible, current-related inhibition of firing in 40 of 59 neurones in the dorsolateral PAG. In 8 of 10 neurones the effect of SIN 1 was significantly reduced after iontophoresis of methylene blue (10-30 nA for 2.7-5 min). The inhibition took up to 7 min to develop and lasted for up to 13 min. Inhibitory responses to GABA were not affected by methylene blue. Iontophoresis of SNOG also inhibited ongoing activity of 18 of 24 neurones tested in the PAG. The experiments demonstrate firstly that NO donors can be used in vivo to deliver NO in the vicinity of neurones by iontophoresis from micropipettes. Secondly, NO appears to inhibit neuronal activity within the PAG.