OBJECTIVE To investigate neuroanatomical characteristics of the primary sensory afferent and the motor neurons coming from and innervating acupoint "Chengshan" (BL 57) area in the rat by using cholera toxin subunit B conjugated with Alexa Fluor 488 (CTB-Alexa 488), a new generation of fluorescent neural tracing reagent. METHODS Four male SD rats were used in the present study. Under anesthesia, 0.05% CTB-Alexa 488 (5 ML) was injected into the central part of the rear of the hind leg, a corresponding site of "Chengshan" (BL 57) in the human body. After 40-48 surviving hours, the rat's brain, spinal cord and dorsal root ganglia (DRGs) of the lumbar segments (L1-L6) were dissected following perfusion with 4% paraformaldehyde, cut into sections and observed under fluorescent microscope equipped with a digital camera. The neurons labeled by CTB-Alexa 488 were counted. RESULTS All CTB-Alexa 488 labeled neurons appeared in green under fluorescent filters of 450-490 and were located ipsilaterally on the injection side. The labeled primary sensory neurons were found in the DRGs at L4 (11 neurons) and L5 (35 neurons). Among them, 29 neurons (63.04%) were bigger, with their cell body diameters being 35-50 microm and 17 (36.96%) smaller, with their body diameters being lower than 35 microm. The labeled motor neurons were found to distribute in the mediolateral portion of lamina IX, forming a longitudinal column from L4 to L5. Of the observed 316 motor neurons, 259 (81.96%) belong to alpha type with their body diameters being 25-40 microm and 57 (18.04%) to gamma type with their body diameters being lower than 25 microm. CONCLUSION The CTB-Alexa 488-labeled primary sensory and motor neurons innervating acupoint "Cheng-shan" (BL 57) distribute in the DRGs of L4-L5. The present fluorescent tracing technique may be quite useful for investigating the neural characteristics of acupoints.