The activity of nitric oxide synthase (NOS) was measured in homogenates from cortex, striatum, hippocampus, cerebellum, pons, thalamus and midbrain of the brain of newborn piglets and the effects of hypoxia and posthypoxic period on this activity was evaluated. The control activities were 19.7, 31.5, 26.8, 16.7, 33.6, 19.3 and 39.4 pmole/mg protein/min, respectively. One hour of hypoxia (decrease cortical oxygen pressure from 46 ± 3 Torr to 8± 2 Torr; p< 0.001) resulted in statistically significant decreases in the activity of NOS in every region of the brain except the cortex. By two hours of reoxygenation following such a hypoxic episode, the NOS activities increased to above control levels in all regions of the brain, but this increase was statistically significant compared to control only in thalamus. Since hypoxia induced the greatest decrease in NOS activity in the cerebellum, the kinetic constants of the enzyme were measured in homogenates from this region of brain. The decreased activity following the hypoxic episode was associated with an approximately four-fold increase in the KM for arginine (from 16.3 ±3.2 μM (control) to 70 ± 11 μM, p<0.005) with no significant change in maximal rate (Vmax). The data show that after a period of hypoxia the NOS activity in the brain of newborn piglets is strongly inhibited. This should lead to marked disturbances in metabolism of NO and the metabolic systems it influences, particularly as hypoxia progresses and during the early phase of reoxygenation (funded by NS-31465).