Objective: To compare the effects of autologous equine serum (AES) and autologous conditioned serum (ACS) on equine articular chondrocyte metabolism when stimulated with recombinant human (rh) interleukin (IL)-1β. Sample: Articular cartilage and nonconditioned and conditioned serum from 6 young adult horses. Procedures: Cartilage samples were digested, and chondrocytes were isolated and formed into pellets. Chondrocyte pellets were treated with each of the following: 10% AES, 10% AES and rhIL-1β, 20% AES and rhIL-1β, 10% ACS and rhIL-1β, and 20% ACS and rhIL-1β, and various effects of these treatments were measured. Results: Recombinant human IL-1β treatment led to a decrease in chondrocyte glycosaminoglycan synthesis and collagen II mRNA expression and an increase in medium matrix metalloproteinase-3 activity and cyclooxygenase-2 mRNA expression. When results of ACS and rhIL-1β treatment were compared with those of AES and rhIL-1β treatment, no difference was evident in glycosaminoglycan release, total glycosaminoglycan concentration, total DNA content, or matrix metalloproteinase-3 activity. A significant increase was found in chondrocyte glycosaminoglycan synthesis with 20% AES and rhIL-1β versus 10% ACS and rhIL-1β. The medium from ACS and rhIL-1β treatment had a higher concentration of IL-1β receptor antagonist, compared with medium from AES and rhIL-1β treatment. Treatment with 20% ACS and rhIL-1β resulted in a higher medium insulin-like growth factor-I concentration than did treatment with 10% AES and rhIL-1β. No difference in mRNA expression was found between ACS and rhIL-1β treatment and AES and rhIL-1β treatment. Conclusions and Clinical Relevance: Minimal beneficial effects of ACS treatment on proteoglycan matrix metabolism in equine chonrocytes were evident, compared with the effects of AES treatment.