Investigation of novel experimental application systems for growth factors or other bioactive substances in tissue engineering is often limited by high costs of substances and would benefit from a defined and easily controllable model tissue system. Herein, we demonstrate a potential three-dimensional in vitro system using engineered cartilage as a model tissue and readily available insulin as a model drug. Previously it has been shown that insulin-like growth factor-I (IGF-I) has profound effects on tissue-engineered cartilage in vitro. Insulin is known to bind to the IGF-I receptor and to elicit significant responses in cartilage. In this study, bovine articular chondrocytes were seeded onto biodegradable polyglycolic acid (PGA) scaffolds and cultured for up to 7 weeks. Exogenous insulin (0.05-50 μg/ml) increased the growth rate and the glycosaminoglycan fraction of tissue-engineered cartilage, decreased the cell number in the tissue constructs, and improved the morphological appearance, with 2.5 μg/ml being the most favorable concentration. The observed effects of insulin were similar to effects of IGF-I (0.05 μg/ml) and were in agreement with the reported binding constants of IGF-I and insulin at the IGF-I receptor. Besides the possibility to employ insulin as a potent substance to improve tissue-engineered cartilage, the presented easily controllable in vitro system may be used in the future to evaluate experimental growth factor application devices using economically favorable insulin as a model protein.