Objective The combination of chondrocytes and mononuclear fraction (MNF) cells might solve the expansion induced dedifferentiation problem of reimplanted cells in autologous chondrocytes implantation as sufficient cells would be available for direct, one-stage, implantation. Earlier in vitro work already showed a positive stimulation of cartilage specific matrix production when chondrocytes and MNF cells were combined. Therefore, this study aimed to evaluate cartilage regeneration using a one-stage procedure combining MNF cells and primary chondrocytes for the treatment of focal cartilage lesions in goats compared to microfracture treatment. Design Freshly created focal cartilage defects were treated with either a combination of chondrocytes and MNF cells embedded in fibrin glue or microfracture treatment. After 6 months follow-up local regeneration as well as the general joint cartilage health were evaluated using validated scores and biochemical assays. Results Macroscopic (p=0.015) scores for the cartilage surface at the treated defect were, after 6 months, significantly higher for the chondrocyteMNF treatment compared to microfracture-treated defects, but microscopic scores were not (p=0.067). The articulating cartilage showed more (p=0.005) degeneration following microfracture treatment compared to chondrocyte-MNF treatment. Biochemical GAG evaluation did not reveal differences between the treatments. Both treatments had resulted in a slight to moderate cartilage degeneration at other locations in the joint. Conclusion In conclusion, treatment of focal articular cartilage lesions in goats using a combination of mononuclear fraction cells from bone marrow and unexpanded chondrocytes leads to better macroscopic regeneration compared to microfracture, however needs further fine-tuning to decrease the negative influence on other joint compartments.