This communication describes a replating technique for the separation of the chondrogenic and non-chondrogenic cells from stage-24 chick limb bud mesenchymal cell cultures by means of sequential digestion with collagenase. Four sub-populations of cells were obtained: The first consisted solely of non-chondrocytes and the next three were progressively enriched in chondrocytes. In addition to morphological differences, the four cell populations differed from each other in their rates of incorporation of sulfate into macromolecular material which were roughly proportional to the percentage of chondrocytes. The chondrocytes and non-chondrocytes no longer exhibited a density dependence of phenotype. In addition, the normal multilayered nodular morphology associated with cartilage development was not observed. These isolated cells have been used as starting material for detailed biochemical studies. Together, these studies indicate that the expressional program governing biosynthetic changes in chondrocytes is not controlled by the extracellular matrix.