The development of mechanisms of neurotransmitter release is an important component in the formation of functional synaptic connections. Synaptic neurotransmitter release can be modulated by nitric oxide, a compound shown to have a variety of physiologic functions in the nervous system. The goal of this study was to determine whether, during synaptic maturation, nitric oxide is capable of affecting exocytosis of synaptic vesicles, and to compare its effects with those elicited by strongly depolarizing stimuli. To address these questions we examined vesicle release from large numbers of individual synapses of hippocampal neurons between five and 13 days in culture. Synaptic vesicles were labelled by uptake of the styrylpyridinium dye N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43) and their release was monitored by fluorescence imaging. Across populations of developing synapses, there was a good correspondence between FM1-43 staining and synapsin immunocytochemistry. A marked heterogeneity was observed in the ability to release vesicles both after potassium and nitric oxide stimulation. In less mature populations of synapses, the rate of potassium- and nitric oxide-induced exocytosis gradually increased, while at later stages nitric oxide-induced responses levelled off and potassium-induced responses continued to rise. Application of nitric oxide donors did not trigger any detectable changes in intracellular calcium. Combined immunocytochemical analysis of cultured hippocampal neurons for neuronal nitric oxide synthase and synapsin revealed that nitric oxide synthase was present within neurites of cultured hippocampal neurons, largely distributed in a bead-like pattern which partially overlapped presynaptic sites. Stimulation of the N-methyl-D-aspartate receptor while blocking propagation of action potentials with tetrodotoxin resulted in exocytosis from numerous individually resolved sites. Preincubation of neurons with an nitric oxide synthase inhibitor or addition of an nitric oxide scavenger eliminated these responses indicating a role for nitric oxide in N-methyl-D-aspartate-stimulated exocytosis. Using fluorescence imaging of individually resolved synaptic sites, we provide direct evidence for an effect of nitric oxide on vesicular neurotransmitter release in intact neurons. Nitric oxide is capable to produce this effect at all stages of synaptic development and acts independently of calcium influx. We show that nitric oxide synthase is present at synaptic sites and endogenously produced nitric oxide is sufficient to cause exocytosis. Taken together, these experiments suggest a possible role for nitric oxide in calcium-independent transmitter release in populations of synapses at all stages of maturation.