Abstract: Objective To establish the primary culture method of hippocampal neurons of new-born rats in vitro, and observe the morphological characteristics at different developmental and differential stages. Methods After separated from Wistar rats born within 24 hours, the hippocampus was digested and the cells were seeded in sterile flask. 1h later, the neurons were transferred and re-seeded on cover glasses, which were coated with poly-L-lysine previously. The neurons were identified by immunocytochemistry with polyclonal antibody to neuron specific enolase (NSE). The morphological characteristic was observed under phase-contrast microscope at different time after seeding. Results A large number of hippocampal neurons began to adhere to the cover glasses 12-24 hours later. They showed different shapes-shuttle, triangle, pyramidal, or nonregular after clinging to the plate. Their processes connected into nets and were different in length and thickness, usually with one of them longer than the others. They were well developed on the 7th-10th day and could survive as long as 28 days after seeding. Immunocytochemistry of NSE indicated high purity of neurons. Conclusions: The culture method of new-born rat hippocampal neurons in vitro is successful and can be used for further study.