Cartilage cells are normally studied under atmospheric pressure conditions and without loading. However, since cartilage exists in a condition of reduced oxygen and intermittent hydrostatic pressure we hypothesized lower partial oxygen pressures (PO₂) and different intermittent hydrostatic pressures (IHP) would increase articular chondrocyte proliferation and matrix production and to stabilize chondrocyte phenotype in vitro. Monolayers of adult bovine articular chondrocytes were cultured under 5% or 21% PO₂ in combination with IHP (0.2 MPa amplitude, frequencies 5/5 s=0.1 Hz, 30/2 or 2/30 min on/off loading). We measured proliferation (³H-thymidine incorporation) and collagen secretion (protein-binding assay, collagen type II-ELISA and immunocytochemical staining of pericellular collagen types I, II and IX). Reduced PO₂ stimulated proliferation and collagen type II and IX secretion of chondrocytes in comparison to 21% PO₂. Additionally, collagen type I expression was delayed by low PO₂, indicating a stabilization of the cell phenotype. IHP 5/5 s and 30/2 min inhibited proliferation but increased collagen secretion (pericellular collagen type IX was decreased). IHP 30/2 min delayed first expression of collagen type I. In contrast, IHP 2/30 min increased proliferation, but lowered collagen expression. All stimulating or inhibiting effects of PO₂ and IHP were additive and vice versa. Reduced PO₂ and different settings of IHP increased proliferation, collagen secretion, and phenotype stability of chondrocytes. The oxygen- and IHP-induced effects were additive, suggesting that a combination of these parameters might be a useful tool in cartilage tissue engineering.