The ideal in vitro system for investigating the regulation of cartilage formation and maintenance would allow for three-dimensional tissue growth, a wide range of biochemical interventions, and non-destructive evaluation. We have developed a hollow fiber bioreactor (HFBR) system which meets these criteria. After injection with embryonic chick sternal chondrocytes, neocartilage is elaborated around the hollow fibers, reaching a thickness of up to a millimeter after four weeks of growth. This process was monitored over time with nuclear magnetic resonance (NMR) microimaging and correlative biochemical and histologic analyses. Tissue volume and cellularity increased greatly during development. This was accompanied by changes in magnetic resonance properties consistent with increased macromolecular content. Further, tissue heterogeneity, observed as regional variations in cell size in histologic sections, was also observed in quantitative NMR images.