Endothelial nitric oxide synthase (eNOS) has been implicated in various brain and peripheral pathologies such as renal failure, heart failure or stroke. Consequently, the mortality rate of aged eNOS knockout mice (eNOS-/-) was higher than that of age-matched (18-22 months old) controls. Only seven of the original 14 eNOS-/- animals that participated in the study reached the age of 18 months or older, whereas no control mice died during this life span. In order to assess the behavioral and neurochemical consequences of chronic eNOS deficiency we examined whether the surviving aged eNOS-/- mice showed changes in terms of motor, emotional, exploratory and neurochemical parameters. Aged eNOS-/- mice showed reduced exploratory activity in the open-field with no habituation observable neither within sessions nor after repeated exposures. Pole test performance of eNOS-/- mice was comparable to controls. In the elevated plus-maze eNOS-/- mice did not differ from controls in terms of time spent in and entries into arms, but showed less locomotion on the open arms. The most prominent neurochemical alterations in the forebrains of aged eNOS-/- mice were: (a) increased acetylcholine levels in the neostriatum; (b) decreased noradrenaline concentrations in the ventral striatum; and (c) lower serotonin levels in the frontal cortex and ventral striatum. The present findings suggest that mice which survived chronic eNOS-deficiency into old age, show some behavioral and neurochemical phenotypes distinct from adult eNOS-/- mice.