The relationship between enkephalinase inhibition by thiorphan in vivo and analgesic activity in nociceptive tests was studied. The analgesic activity of thiorphan in various nociceptive tests was compared with that of a narcotic analgesic, morphine and antipyretic analgesic, antipyrine. Tail-flick test revealed that thiorphan applied intracerebroventricularly (i.c.v.) or intraperitoneally (i.p.) in rats markedly potentiated the analgesic activity of [D-Ala2, Met5]-enkephalin administered i.c.v. The amount of thiorphan in the mouse brain and the fragments of Met-enkephalin degraded by brain homogenate were assayed after intraperitoneal administration of 300 mg/kg thiorphan. The concentration of thiorphan in the brain arised to 18.2 +/- 2.4 nmol/g brain 30 min after intraperitoneal administration of thiorphan and then it quickly disappeared from the brain. As to the fragments of Met-enkephalin degraded by brain homogenate after i.p. administration of thiorphan, the concentration of tyrosine and tyrosyl-glycine (Tyr-Gly) was the same as that of the vehicle control, whereas only the amount of tyrosyl-glycyl-glycine (Tyr-Gly-Gly) decreased to 21.5% of the control value after 30 min, and then it recovered to 75% after 180 min. Thus, thiorphan inhibited enkephalinase activity alone demonstrating selective activity. Thiorphan at doses of 30-300 mg/kg demonstrated analgesic activity in the nociceptive tests of acetic acid writhing, hot-plate and tail-flick, whereas it did not have any activity in the tail-pinch test. Morphine showed analgesic activity in the four nociceptive tests employed. Antipyrine showed analgesic activity in three nociceptive tests but not in the tail-flick test. The dose response curves for morphine and antipyrine were parallel. The slope of the dose response curve for thiorphan, however, was shallower than those for two reference analgesics used. The role of main enkephalin degrading enzymes in the brain was discussed with respect to the analgesic action of thiorphan and its concentration in the brain.