OBJECTIVE To investigate the cytotoxic effects of 2 different concentrations of buprenorphine and compare them with bupivacaine and morphine on healthy equine chondrocytes in vitro. SAMPLE Primary cultured equine articular chondrocytes from 3 healthy adult horses.PROCEDURES Chondrocytes were exposed for 0 and 2 hours to the following treatments: media (CON; negative control); bupivacaine at 2.2 mg/mL (BUPI; positive control); morphine at 2.85 mg/mL (MOR); buprenorphine at 0.12 mg/mL (HBUPRE); or buprenorphine at 0.05 mg/mL (LBUPRE). Chondrocyte viability was assessed using live/dead stain- ing, water-soluble tetrazolium salt-8 (WST-8) cytotoxic assay, LDH assay, and flow cytometry. All continuous vari- ables were evaluated with a mixed ANOVA with treatment, time, and their interactions as the fixed effects and each horse as the random effect.RESULTS Buprenorphine showed a concentration-dependent chondrotoxic effect. The viability of chondrocytes was signifi- cantly decreased with exposure to HBUPRE and BUPI compared to CON, MOR, and LBUPRE.CLINICAL RELEVANCE Negligible chondrotoxic effects were observed in healthy cultured equine chondrocytes exposed to 0.05 mg/mL of buprenorphine, whereas higher concentrations (0.12 mg/mL) showed a marked cytotoxic effect. Based on these results, low concentrations of buprenorphine appear to be safe for intra-articular administration. Further evaluation of this dose in vivo is needed before recommending its clinical use.