Neuronal NO synthase (nNOS) was discovered recently to interact specifically with the protein PIN (protein inhibitor of nNOS) [Jaffrey, S.R. and Snyder, S.H. (1996) Science 274, 774^777]. We have studied the effects on pure NOS enzymes of the same GST-tagged PIN used in the original paper. Unexpectedly, all NOS isoenzymes were inhibited. The IC SH for nNOS was 18 þ 6 W WM GST-PIN with 63 nM nNOS after 30 min at 37³C. Uncoupled NADPH oxidation was inhibited similarly, whereas cytochrome c reductase activity, the K w for L-arginine, and dimerization were unaffected. We reconsider the physiological role of PIN in the light of these results.z 1998 Federation of European Biochemical Societies.