During development, limb-bud mesenchymal cells carry out complex spatiotemporal-patterns of growth and differentiation. Tissue and organ culture facilitate analysis of environmental influences on these cell behaviors, allowing their partial dissection into exogenous and endogenous components. Two factors that complicate such in vitro analyses are the heterogeneity of the cultured cells and imprecise knowledge of culture medium composition. Limb mesenchyme comprises a heterogenous cell population with important regional differences in cell type. Dividing the limb into subregions helps limit the cellular heterogeneity and using chemically defined, serum-free medium alloys concerns about medium composition. In the present study, mesenchyme from different regions along the anteroposterior and proximodistal axes of stage 21-22 and stage 23-24 chick wing buds was grown in high-density microtiter cultures in chemically defined and in serum-containing medium. Four-day cultures of the various regions were compared in terms of culture morphology and the accumulation of Alcian blue-positive cartilage matrix and DNA. The results demonstrate stage- and region-dependent differences in the in vitro growth, differentiation, and responsiveness of these cells. For example, mesenchyme from the distal anterior region of the wing bud exhibited lower intrinsic chondrogenic capacity and greater responsiveness to serum than other regions. Patterns of in vitro chondrogenesis also suggest that, at the stages examined, distal wing-bud mesenchyme may be less homogeneous than has been believed. A case is made for the suitability of serum-free medium for future in vitro studies of chick limb-bud mesenchyme. The results are considered in relation to the process of limb development and regional expression of pattern-related genes.