We aimed to investigate freshly isolated compared with culture-expanded chondrocytes with respect to early regenerative response, cytokine production and cartilage formation in response to four commonly used biomaterials. Materials & methods: Chondrocytes were both directly and after expansion to passage 2, incorporated into four biomaterials: Polyactive™, Beriplast®, HyStem® and a type II collagen gel. Early cartilage matrix gene expression, cytokine production and glycosaminoglycan (GAG) and DNA content in response to these biomaterials were evaluated. Results: HyStem induced more GAG production, compared with all other biomaterials (p ≤ 0.001). Nonexpanded cells did not always produce more GAGs than expanded chondrocytes, as this was biomaterial-dependent. Cytokine production and early gene expression were not predictive for final regeneration. Conclusion: For chondrocyte-based cartilage treatments, the biomaterial best supporting cartilage matrix production will depend on the chondrocyte differentiation state and cannot be predicted from early gene expression or cytokine profile.