Objective To research the possible analgesic mechanism of Pain-Relieving Formula Ⅰ Tincture for external use. Methods Twenty Wistar rats were randomized into the treatment group and control group

with 10 in each. Pain-Relieving Formula Ⅰ Tincture and 75% alcohol was applied topically on the hind feet and entire thighs of rats in the treatment group and control group respectively

0.3 ml per rat

20 min once for 6 times continously. The right hind feet of rats were injected with 5% formalin solution 0.1 ml and the paw thickness and perimeters were measured 30 min after the last administration. The contents of substance-P （SP） and β-endorphin in spinal cord segments L4-5 and brain were observed by immunohistochemistry. Results The hind paw thickness and perimeters were significantly increased in both groups after proinflammation （P<0.05）. The hind paw thickness and perimeters in the treatment group were lower than those in the control group after proinflammation （P<0.05）. The content of β-endorphin in spinal cord and brain in the treatment group was higher than that in the control group （P<0.05）. The content of SP in spinal cord in the treatment group was lower than that in the control group （P<0.05）. There was no significant difference between groups in the content of SP in the brain tissue （P>0.05）. Conclusion Pain-Relieving Formula Ⅰ Tincture has a better anti-inflammatory and analgesic effect. Its analgesic mechanism may be related to increasing the content of antinociceptive neurotransmitter β-endorphin in the brain and spinal cord and decreasing the content of nociceptive neurotransmitter SP.