The aim of this study was to investigate the effects of serum and compressive dynamic loading on the cartilaginous matrix spatiotemporal distribution around chondrocytes in vitro. Murine chondrocytes suspended in agarose were cultured in serum-free media or in varying concentrations of serum with or without compressive dynamic loading. Gene expression was assayed by quantitative polymerase chain reaction. Immunohistochemistry was performed for type II collagen and type VI collagen, aggrecan, or cartilage oligomeric matrix protein (COMP) to study the effect of serum and dynamic loading on the spatiotemporal distribution of cartilage matrix components. Chondrocytes in serum-free culture exhibited negligible differences in type II collagen, aggrecan, and COMP mRNA expression levels over 15 days of cultivation. However, higher serum concentrations decreased matrix gene expression. Expression of the matrix metalloproteinases (MMP)-3 and MMP-13 mRNA increased over time in serum-free or reduced serum levels, but was significantly suppressed in 10% fetal bovine serum (FBS). Compressive loading significantly stimulated MMP-3 expression on days 7 and 15. Immunohistochemical analysis demonstrated that maximum pericellular matrix deposition was achieved in 10% FBS culture in the absence of compressive loading. The pericellular distribution of type II and VI collagens, aggrecan, and COMP proteins tended to be more co-localized in the pericellular region from day 9 to day 21; compressive loading helped promote this co-localization of matrix proteins. The results of this study suggest that the quantity, quality, and spatial distribution of cartilaginous matrix can be altered by serum concentrations and compressive loading.