The objective of this study was to study the matrix production and phenotype stability of articular chondrocytes cultured on collagen I/III membranes (CM) under the influence of low oxygen tension (Po(2)). Primary bovine and osteoarthritic human chondrocytes were cultured for 2 weeks under 5-21% Po(2) on CM, in alginate, or as monolayers. Dedifferentiated cells were produced by 2-week monolayer culture under 21% Po(2). Collagen (Coll) type II and I expression was demonstrated immunohistochemically, by Western blotting (Coll II), and by semiquantitative RT-PCR; proteoglycan synthesis was demonstrated histochemically (toluidine blue); and biosynthetic activity was indicated by radiolabel incorporation ([(3)H]proline and [(35)S]sulfate). Bovine chondrocytes on CM showed an increase in Coll II expression and proteoglycan synthesis under low Po(2) conditions, whereas Coll I decreased. This oxygen-dependent phenotype-stabilizing effect was even more pronounced in alginate cultures. Biosynthesis of bovine and human chondrocytes was also increased by low Po(2), except for proline incorporation, which decreased in bovine CM cultures (low-oxygen effects were significantly higher in alginate than in CM cultures). Dedifferentiated chondrocytes reexpressed Coll II protein when cultured under low Po(2) on CM or in alginate only, but not under high Po(2) or in monolayer culture. We conclude that CM and, even more, alginate foster phenotype stability and cartilage-specific matrix production of bovine chondrocytes, especially when cultured under in vivo-like oxygen conditions.