The involvement of spinal serotonergic system in testosterone influence on formalin-induced pain was studied in male rats. Four weeks after castration, there was an analgesia in the late phase of formalin test that was reversed by intraperitoneal injection of testosterone enanthate (1 mg/kg) for 3 days. Flutamide (testosterone antagonist) produce analgesia in the late phase on intraperitoneal (10 mg/kg, IP) and intrathecal (60 microg/rat, IT) injections, but not on intracerebroventricular (60 microg/rat, ICV) administration. The antinociceptive effect of castration and IP flutamide (10 mg/kg) was abolished by pretreatment with 5,7-dihydroxytryptamine (5,7-DHT, 100 microg/rat, IT). IT-administered 5-HT (100 microg/rat) produced analgesia in the early and late phase of formalin test. Microdialysis sampling was used to characterize the extracellular concentration of 5-hydroxytryptamine (5-HT, serotonin) in the dorsal horn of the lumbar spinal cord. This technique demonstrated that levels of 5-HT were increased in 4-week castrated and IP flutamide (10 mg/kg) injected rats. The results may indicate that the analgesia produced by castration and flutamide administration is mediated through functional alteration in spinal cord serotonergic system.