Neuronal nitric oxide synthase (nNOS) is expressed most densely in two types of cortical interneurons. Large nitric oxide (LNOS) interneurons express dense somatostatin- and neuropeptide Y-immunoreactivity, whereas small (SNOS) interneurons are calbindin-immunoreactive. We used double labeling methods in several areas of the monkey cerebral cortex to determine how precisely these neurochemical labels define the two types of nNOS interneurons. The LNOS cells were essentially always (>99%) immunoreactive for somatostatin and neuropeptide Y, but did not express calbindin. The LNOS cells comprised about 30% of the somatostatin cells and about 60% of the neuropeptide Y cells. The SNOS cells were nearly always (87-98%) calbindin-immunoreactive, and were rarely or never labeled with antibodies to somatostatin or neuropeptide Y. The SNOS cells accounted for about 20% of all of the calbindin cells. The findings demonstrate that the two types of nNOS cells can be distinguished by antibodies to calbindin, somatostatin and neuropeptide Y, but none of these markers is found exclusively in nNOS cells. Nevertheless, neuropeptide Y-immunoreactivity provides a useful marker for LNOS cells, because it is very dense in these cells and only light in the interneurons that lack nNOS.