Subplate neurons are early-generated neurons that project into the overlying neocortex and are required for the formation of ocular dominance columns. A subset of subplate neurons express nitric oxide synthase (NOS) and produce nitric oxide (NO), a neuronal messenger thought to be involved in adult hippocampal synaptic plasticity and also in the establishment of certain specific connections during visual system development. Here, we examine whether the NOS-containing subplate neurons are involved in ocular dominance column formation in the ferret visual system. Ocular dominance columns form in ferrets between postnatal day 35 (P35) and P60. NOS expression in the visual subplate is low at birth, increases to a maximum at the onset of ocular dominance column formation, and falls thereafter. Nevertheless, blockade of NOS with daily injections of nitroarginine from P14 to P56 fails to prevent the formation of ocular dominance columns, although NOS activity is reduced by >98%. To test further a requirement for NOS in the patterning of connections during CNS development, we examined the cortical barrels in the somatosensory system of mice carrying targeted disruptions of NOS that also received injections of nitroarginine; cortical barrels formed normally in these animals. In addition, barrel field plasticity induced by whisker ablation at birth was normal in nitroarginine-injected NOS knock-out mice. Thus, despite the dynamic regulation of NOS in subplate neurons, NO is unlikely to be essential for the patterning of thalamocortical connections either in visual or somatosensory systems.