To determine whether low oxygen (O(2)) tension during expansion affects the matrix density, as well as quantity, of cartilage formed, and to determine whether application of low O(2) tension during incubation periods alone is sufficient to modulate chondrogenic expression, rabbit chondrocytes expanded at either 21% O(2) or 5% O(2) were analyzed for glycosaminoglycan (GAG) and DNA content, total collagen, and gene expression during expansion and postexpansion aggregate cultures. When cultured as aggregates at 21% O(2), chondrocytes expanded at 5% O(2) produced cartilage aggregates that contained more total GAG, GAG per wet weight, GAG per DNA, and total collagen than chondrocytes expanded at 21% O(2). Less of an effect on GAG and collagen content was observed when aggregate culture was performed at 5% O(2). Real-time polymerase chain reaction analysis of COL2A1 expression showed upregulated levels of type IIA (an early marker) and IIB (a late marker) during expansion and elevated levels of type IIB during aggregate culture in chondrocytes expanded in low O(2). The application of low O(2) tension during incubation periods of chondrocyte expansion enhances the ultimate cartilage matrix density and quantity, and this enhancement can be achieved through the use of an O(2) control incubator.