During endochondral ossification, small rapidly proliferating chondrocytes mature into flattened disc-shaped cells and then into large round hypertrophic cells. These morphological changes are accompanied by a decrease in the rate of cell proliferation. Type X collagen synthesis is initiated during chondrocyte maturation and reaches very high levels in the hypertrophic cells. We have analyzed type X collagen gene expression in chick embryo vertebral chondrocytes that were allowed to mature in monolayer culture and were then switched to suspension culture. The resuspended chondrocytes changed in shape from flat to round and decreased the proliferation rate as they do in vivo. These events were accompanied by a rapid, dramatic increase in type X collagen gene expression at the levels of transcription, steady-state mRNA and protein synthesis, as well as an increase in the number of cells producing type X collagen. The amount of type X collagen gene expression in resuspended chondrocytes was comparable to that in mineralizing cartilage in vivo. These results indicate that events accompanying the switch from monolayer to suspension culture (for example, the change from a flat to a round shape and/or the decrease in proliferation rate) may play a role in the induction of type X collagen gene expression during chondrocyte maturation. Thus we have developed an in vitro system that appears to mimic the events occurring during in vivo chondrocyte maturation. This in vitro model may provide an ideal system for further examination of the parameters regulating chondrocyte maturation and type X collagen gene expression.