AIM: To isolate and culture the adipose-derived stem cells (ADSCs) from guinea pig so as to create bases for the experimental study on treating sensorineural deafness by ADSCs transplantation. METHODS: The experiment was carried out between December 2005 and March 2006 in the Open and Key Laboratory of Clinical Medicine at Henan Universities. The inguinal fat pads were excised from adult male guinea pig weighing 500-750 g. A great quantity of adhesive and natant blood cells were observed under inserted microscope and phosphate buffer was used to wash natant cells repeatedly. Then the cells were cultured in DMEM with 10% fetal bovine serum. Before passage, the cells were washed by a small quantity of phosphate buffer and then cultured in 0.25% trypsin and 0.02% EDTA (2 mL). Then most of the ADSCs presented cytoplasm recovery and round shape, followed by digestion of 2 mL DMEM with feral bovine serum. Cellular suspension was collected to count the cells that were incubated at the density of 2?104/cm2. The morphology of ADSCs was observed constantly. Growth curve of ADSCs at passages 1, 3, 10 were recorded and detected by MTT. Surface markers of ADSCs were detected by flow cytometry. RESULTS: ①The result of primary culture showed that ADSCs at day 2 began to adhesion and more than 90 % of ADSCs at day 7 were approximately fusiform or fibroblast-shaped.②MTT detection showed that ADSCs had the capability to proliferate successively, especially at passage 1, 2, 3. But ADSCs at passage 10 presented decreasing proliferation. After digestion, the cells at passages 1, 3, 10 experienced the latent period (24-48 hours), logarithmic phase (3-5 days) and growth platform phase.③CD105 and CD44 were positive by flow cytometry, whereas CD34 was negative. CONCLUSION: Due to stable growth and rapid proliferation, the ADSCs from guinea pig can be used as the donor cells in the treatment of sensorineural deafness by stem cell transplantation.