The effect of multiple intracerebroventricular (i.c.v.) injections of [D-Pen2, D-Pen5]enkephalin (DPDPE), a delta-opioid receptor agonist, on the activity of nitric oxide synthase (NOS) was determined in the brain regions and spinal cord of the mouse. Male Swiss Webster mice were injected twice daily with DPDPE (20 micrograms/mouse, i.c.v.) or its vehicle for 4 days. This procedure has previously been shown to induce tolerance to the antinociceptive actions of DPDPE in mice. On day 5, the animals treated with DPDPE were either sacrificed 20 min after an i.c.v. injection of DPDPE (tolerant) or without any injection (abstinent i.e., 16 h after the last injection of DPDPE). NOS activity in brain regions (cortex, striatum, hippocampus, midbrain, pons/medulla, hypothalamus and cerebellum) and spinal cord was determined by the rate of conversion of arginine into citrulline. Tolerance to DPDPE was associated increases in NOS activity in midbrain (49%) and pons/medulla (32%) and decreases in cerebellum (28%) and spinal cord (44%). However, NOS activity was unchanged in the cortex, corpus striatum, hippocampus and hypothalamus. On the other hand, during abstinence from DPDPE, NOS activity increased in midbrain (84%) and hypothalamus (35%) but decreased in cerebral cortex (27%) cerebellum (27%) and spinal cord (20%). NOS activity was unchanged in the corpus striatum, hippocampus and pons/medulla. Previous studies from this laboratory had demonstrated that chronic administration of mu- and kappa-opioid receptor agonists results in increases NOS activity in certain brain regions and that tolerance to mu- and kappa-, but not to delta-opioid receptor agonists, is attenuated by NOS inhibitors. The present studies, for the first time, demonstrate decreases in NOS activity in certain brain regions and spinal cord of mice treated chronically with delta-opioid receptor agonist. Furthermore, these findings may explain the inability of NOS inhibitors to attenuate tolerance to DPDPE in mice.