Objective To obtain highly pure cultured Sertoli cells from rat testis and identificate cultured cells by in situ hybridization for ABP mRNA. Methods Testes from 18-22 day old SD rats were removed and decapsulated,then chopped and sequentially digested with three enzymes at 37℃: first with 0.25% trypsin for 15 minutes,then with 0.1% hyaluronidase for 30 minutes,at last with 0.1% collagenase V for 2-3 hours.The isolated cells were incubated at 32℃ in a humidified atmosphere of 5% CO2.To increase the purity of the Sertoli cells, the cultured cells were subjected to hypotonic shock(treatment) with 20 mmol Tris-HCl after 48 hours of incubation.After a week’s incubation,the cultured cells were identificed by HE staining,AO fluorescence staining,Feulgen staining,and in situ hybridization with digoxin-labeled rat ABP cDNA. Results Over 95% of the cultured cells were Sertoli cells.Most of the cultured cells expressed ABP mRNA and their structural features were the same as those of the Sertoli cells identified by other methods.Highly pure Setoli cells can be obstained by the sequentiall digestion with three enzymes and hypotonic shock.Conclusion As a specifical secreted protein secreted Sertoli cells in testes,ABP mRNA detected by in situ hybridization is a new, specific,effective identification method of Sertoli cells from testes.Meanwhile as an