Under conditions in which NG-nitro-L-arginine (NOArg) treatment prevents morphine tolerance, NOArg induces a slow progressive inhibition of nitric oxide synthase (NOS), starting at approx. 20% after a single treatment and increasing to approx. 65% after 10 days. Studies designed to examine potential changes in NOS levels with chronic morphine administration reveal no change. Total NOS activity in both brainstem and cerebellum homogenates is unchanged, as are levels of NOS mRNA in a variety of brain regions. L-Arginine, the precursor of nitric oxide (NO), accelerates tolerance when coadministered with morphine and when given alone L-arginine decreases morphine's potency. Administration of L-arginine alone for 3-10 days shifts morphine's dose-response curve over 2-fold to the right while D-arginine is without effect, as is daily administration of L-arginine along with the NOS inhibitor NOArg. Thus, chronic L-arginine induces "tolerance" in opioid naive mice through NOS. Together, our data indicate an important role for NO in the modulation of opioid analgesia.