OBJECTIVE: To compare the chondrotoxicity of bupivacaine, lidocaine, and mepivacaine in equine articular chondrocytes in vitro. STUDY DESIGN: Prospective, experimental study. STUDY MATERIAL: Equine articular chondrocytes. METHODS: Primary cultured equine chondrocytes were exposed to 0.5% bupivacaine, 2% lidocaine, or 2% mepivacaine for 30 or 60 minutes. After treatment, cell viability was evaluated by trypan blue exclusion and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in a dose dependent manner. Apoptosis and necrosis of chondrocytes were analyzed with the double staining of Hoechst 33258 and propidium iodide using fluorescence microscopy, and the results were confirmed using flow cytometry. RESULTS: After 30-minute exposure, trypan blue exclusion assay revealed that cell viability of 0.5% bupivacaine group was 28.73±8.44%, and those of 2% lidocaine and 2% mepivacaine were 66.85±6.03% and 86.27±2.00%, respectively. The viability of chondrocytes after saline treatment was 95.95±2.75%. The results of MTT assay and fluorescence microscopy had similar tendency with trypan blue assay. Each result showed that bupivacaine was the most toxic of the three local anaesthetics. Mepivacaine was less toxic than lidocaine. The results of the viability test suggest that bupivacaine and lidocaine exhibit a marked chondrotoxicity, and that this is mainly due to necrosis rather than apoptosis. CONCLUSIONS AND CLINICAL RELEVANCE: Bupivacaine may induce detrimental chondrotoxicity when administered intra-articularly, especially in patients with joint disease, and we suggest that it should be used cautiously in equine practice. Mepivacaine may be an alternative to both bupivacaine and lidocaine.