Modern tissue engineering strategies comprise three elemental parameters: cells, scaffolds and growth factors. Articular cartilage represents a highly specialized tissue which allows frictionless gliding of corresponding articulating surfaces. As the regenerative potential of cartilage is low, tissue engineering-based strategies for cartilage regeneration represent a huge challenge. Prostaglandins function as regulators in cartilage development and metabolism, especially in growth plate chondrocytes. In this study, it was analyzed if prostaglandin E2 (PGE2) has an effect on the phenotypic differentiation of human chondrocytes cultured in a three-dimensional (3D) gelatin-based scaffold made by directional freezing and subsequent freeze-drying. As a result, it was clearly demonstrated that low doses of PGE2 revealed beneficial effects on the phenotypic differentiation and collagen II expression of human articular chondrocytes in this 3D cell culture system. In conclusion, PGE2 is an interesting candidate for tissue engineering applications since it represents an already well-studied molecule which is available in pharmaceutical quality. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.