The nitric oxide (NO) synthase/cGMP pathway has been studied in vivo in the adult rat hippocampus by monitoring the levels of extracellular cGMP during microdialysis in conscious unrestrained animals. The basal cGMP efflux was concentration-dependently reduced upon local infusion of the NO synthase inhibitor NG-nitro-L-arginine (NARG; 10 microM to 1 mM). The NO donors hydroxylamine and S-nitroso-N-penicillamine, perfused through the dialysis probe at 1 mM, increased by about 200% the extracellular levels of cGMP. The glutamate receptor agonist NMDA (125–500 microM) produced concentration-dependent cGMP responses that were abolished by the selective receptor antagonist D-2-amino-5- phosphonovaleric acid or by NARG. Local perfusion of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM) produced a steady eightfold increase of extracellular cGMP levels. The effect of IBMX was highly sensitive to NARG. The inhibition by NARG of the IBMX-induced cGMP response was reversed when the NO synthase substrate L-arginine was administered. It is concluded that cGMP collected during in vivo microdialysis reflects NO synthase activity in the rat hippocampus. The technique may be utilized to investigate the pathophysiology and the pharmacology of the NO/cGMP pathway in the hippocampus of living animals.