In order to improve the method of separating and cultivating the embryonic rat cerebral neurons in vitro, we compared different digestive enzymes, mechanical separating methods and two different mediums. It was found that highly purified and active embryonic rat cerebral neurons could be harvested by cultivating the neurons in the standard neurobasal medium with B-27 supplement, 10% horse serum, 10% fetal bovine serum and L-Glutamine after separated by digestion with the use of 0.05% trypsin and 0.05% collagenase II.