A subtle mutation in the type II collagen gene COL2A1 was detected in a case of human hypochondrogenesis by using a chondrocyte culture system and PCR-cDNA scanning analysis. Chondrocytes obtained from cartilage biopsies were dedifferentiated and expanded in monolayer culture and then redifferentiated by culture over agarose. Single-strand conformation polymorphism and direct sequencing analysis identified a G----A transition, resulting in a glycine substitution at amino acid 574 of the pro alpha 1(II) collagen triple-helical domain. Morphologic assessment of cartilage-like structures produced in culture and electrophoretic analysis of collagens synthesized by the cultured chondrocytes suggested that the glycine substitution interferes with conversion of type II procollagen to collagen, impairs intracellular transport and secretion of the molecule, and disrupts collagen fibril assembly. This experimental approach has broad implications for the investigation of human chondrodysplasias as well as human chondrocyte biology.