Pre-embedding electron microscopic immunocytochemistry was used to examine the ultrastructure of neurons containing nitric oxide synthase and to evaluate their synaptic relationships with target neurons in the striatum and sensorimotor cerebral cortex. Intense nitric oxide synthase immunoreactivity was found by light and electron microscopy in a type of aspiny neuron scattered in these two regions. The intensity of the labeling was uniform in the soma, dendrites and axon terminals of these neurons. In both forebrain regions, nitric oxide synthase-immunoreactive neurons received synaptic contacts from unlabeled terminals, which were mostly apposed to small-caliber dendrites. The unlabeled symmetric contacts were generally about four times as abundant as the unlabeled asymmetric contacts on the nitric oxide synthase-immunoreactive neurons. Terminals labeled for nitric oxide synthase were filled with synaptic vesicles and were observed to contact unlabeled neurons. Only 54% (in the cerebral cortex) and 44.3% (in the striatum) of the nitric oxide synthase-immunoreactive terminals making apposition with the target structures were observed to form synaptic membrane specializations within the plane of the randomly sampled sections. The most common targets of nitric oxide synthase-immunoreactive terminals were thin dendritic shafts (54% of the immunoreactive terminals in the cortex and 75.7% of the immunoreactive terminals in the striatum), while dendritic spines were a common secondary target (42% of the immunoreactive terminals in the cortex and 20.6% of the immunoreactive terminals in the striatum). The spines contacted by nitric oxide synthase-immunoreactive terminals typically also received an asymmetric synaptic contact from an unlabeled axon terminal. These findings suggest that: (i) nitric oxide synthase-immunoreactive neurons in the cortex and striatum preponderantly receive inhibitory input; (ii) nitric oxide synthase-containing terminals commonly make synaptic contact with target structures in the cortex and striatum; (iii) spines targeted by nitric oxide synthase-containing terminals in the cortex and striatum commonly receive an asymmetric contact as well, which may provide a basis for a synaptic interaction of nitric oxide with excitatory input to individual spines.