Co-culture models have been increasingly used in tissue engineering applications to understand cell–cell interactions and consequently improve regenerative medicine strategies. Aiming at further elucidating cartilage tissue formation, we co-cultured bovine primary chondrocytes (BPCs) with human expanded chondrocytes (HECs), human dermal fibroblasts (HDFs), mouse embryonic stem cells (MESCs), or mouse-3T3 feeder cells (M3T3s) in micromasses. BPCs were either co-cultured (1 : 5 ratio) with all cell types allowing direct cell–cell contacts or as separate micromasses in the same well with HECs. In co-culture groups with direct cell–cell contacts cartilaginous tissue was formed in all experimental groups. In situ hybridization showed that only 16–27% of the cells expressed type II collagen mRNA. Corresponding with the fact that micromasses consisted for approximately 20% only of BPCs, the amount of GAG was similar between 100% BPC micromass and the co-culture groups with HECs and HDFs. Therefore, co-culture micromasses support cartilage tissue formation predominantly originating from primary chondrocytes in direct contact with a variety of cell types. These findings potentially could be applied to optimize cell-therapy treatments for cartilage regeneration.