The neurotransmitter functions of nitric oxide are dependent on dynamic regulation of its biosynthetic enzyme, neuronal nitric oxide synthase (nNOS), By means of a yeast two-hybrid screen, a 10-kilodalton protein was identified that physically interacts with and inhibits the activity of nNOS. This inhibitor, designated PIN, appears to be one of the most conserved proteins in nature, showing 92 percent amino acid identity with the nematode and rat homologs,Binding of PIN destabilizes the nNOS dimer, a conformation necessary for activity. These results suggest that PIN may regulate numerous biological processes through its effects on nitric oxide synthase activity.