The establishment of cells with myogenic or chondrogenic potential is temporally and spatially separated from terminal differentiation in the developing chick embryo. Both cell types arise from tissue adjacent to the neural tube and notochord, the paraxial mesoderm. A cell culture system was developed in order to study the maturation and differentiation of myogenic and chondrogenic cells along the length of the paraxial mesoderm at different stages of development. Somite and segmental plate cells obtained from 36- to 52-h (stages 10-15) embryos were plated as a monolayer on substrata of gelatin, fibronectin, or laminin. A substratum of gelatin plus fibronectin was most effective in supporting adhesion and differentiation. Maximal increase in number of cells in somite cultures occurred 24 h earlier than that in segmental plate cultures. Fewer skeletal muscle cells and chondroblasts were present in cultures prepared from progressively more caudal regions of the paraxial mesoderm and from younger embryos. Some cells present within the somites and the rostral two-thirds of the stage 13 segmental plate differentiated without replication after placement in culture. Only the progeny of cells from its caudal third, and from stage 10 somites and segmental plates, differentiated under these conditions. The results suggest that some myogenic and chondrogenic cells obtain the ability to differentiate under these in vitro conditions after stage 10 of development, as they occupy more rostral positions within the segmental plate relative to the addition of cells at its caudal end. Although some stage 13 segmental plate cells form skeletal muscle and cartilage directly after removal from the embryo, differentiation is not observed in ovo until these cells are incorporated into somites, a minimum of 10 h later. Three-dimensional tissue interactions, and/or cell-cell interactions, while not required for segmental plate cells to undergo myogenesis and chondrogenesis, may play a role in regulating the timing of terminal differentiation within the embryo.