OBJECTIVE To study the humoral factor of acupuncture in improving functional activities of the organism. METHODS Neurons of the cerebral cortex from the new born rats were taken to be cultured in a strictly controlled medium. Seven to ten days later, fluorescein-molecular probe Fluo-3AM was used for staining intracellular Ca(2+). Ca(2+) concentration and its changes after application of acupuncture serum were measured by using a laser confocal microscope. Normal serum and acupuncture serum were derived from the normal control rats and the rats undergoing electro-acupuncture of "Baihui (GV 20)", "Zusanli (ST 36)", "Quchi (LI 11)" and "Sanyinjiao (SP 6)" for 2 weeks. RESULTS After application of normal serum, the intracellular Ca(2+) concentration increased strikingly immediately at the beginning, and gradually got stable afterwards. When acupuncture serum was added to the culture fluid, Ca(2+) concentration lowered significantly. CONCLUSIONS These results suggest that bioactive substances in serum collected from rats undergoing acupuncture can affect the intracellular Ca(2+) concentration of the cerebral neurons, and the humoral factor may participate in the effect of acupuncture in regulating functional activities of the organism.