Two constitutively expressed isoforms of nitric oxide synthase (NOS) have been identified, Nos1 and Nos3. Nos1 was originally identified in neuronal cells and Nos3 in endothelial cells. Because the biochemical activity of NOS is developmentally regulated, we tested the hypothesis that protein expression is also developmentally regulated. Antibodies to Nos1 and Nos3 were evaluated for specificity by immunoblotting and then used for immunohistochemistry. In fetal and adult sheep brain homogenates, Nos1 antibodies identified one immunoreactive band of proteins at 155 kDa. The Nos3 antibody detected one immunoreactive band at 145 kDa that comigrated with a reactive band in endothelial cell lystates. Immunoblots of developing neocortex demonstrated that Nos1 was enriched at early gestational ages, whereas Nos3 expression was relatively constant throughout development. By immunohistochemistry, distinct isoform-specific patterns of immunoreactivity were detected. At 60 days, Nos1 immunoreactivity is primarily localized in neuropil, but by midgestation, nonpyramidal neurons are labeled in the cortical plate. Developing neurites are Nos1-positive at 60 and 71 days, decreasing in abundance by 93 days. By 93 days the striatum is fully populated by Nos1-expressing nonprincipal neurons. In hippocampus and subthalamic nucleus, Nos1 immunoreactivity is greatest at 60 and 71 days gestation, decreasing thereafter. Immunoreactivity for Nos3 delineates cerebrovasculature maturation from a primarily radial to a highly complex branching arrangement. Hindbrain structures achieve mature organization of the cerebrovasculature before forebrain. We conclude that constitutive NOS protein expression is developmentally regulated and that distinct isoforms of NOS are regulated differentially during brain development. Expression of Nos3 parallels maturation of the cerebrovasculature, whereas the transient, region- and cell type-dependent enrichment of Nos1 in the developing brain may indicate a temporally and spatially restricted role for this enzyme in the maturation of specific neuronal populations.