In the presence of added flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), both cytosolic and particulate nitric oxide synthase (NOS) activities can be detected in rat brain. Developmental changes of the cytosolic and particulate NOS in rat cerebellum and cerebrum were determined biochemically and immunochemically. Particulate NOS activity in the cerebrum increased during the first week of development, but then decreased and became almost undetectable in adult rats. In contrast, the cytosolic NOS in cerebellum showed low activity in newborns, but then constantly increased reaching an 8-fold higher level in adult rats. The activities of cerebellar particulate and cerebral cytosolic NOS also increased slightly during maturation. Western blot analysis using a polyclonal antibody raised against rat cerebellar cytosolic NOS revealed that the particulate and cytosolic fractions of 1-week-old and adult rat brains contained the same 160 kDa NOS protein. The relative content of the NOS protein correlated well with the relative amount of NOS activity in all brain fractions. These results indicate that distribution of NOS in the rat brain changes during maturation, but the same NOS protein is likely to be responsible for activities in immature and mature brains, cytosolic and particulate fractions. We suggest that nitric oxide might play a role in functional differentiation of the brain. © 1993.