The character of differentiating chondrocytes in growing long bones has been defined by altered expression of a limited number of genes. To expand this set we have applied differential display to identify genes expressed in either mineralizing or nonmineralizing chondrocytes. One such gene, Band 17, has the following characteristics: (1) Band 17 expression is predominantly found in cartilage destined for mineralization. Band 17 mRNA is undetectable in articular cartilage and undetectable or weak in all other tissues tested. (2) Band 17 expression is spatially restricted to the lower proliferative/upper hypertrophic zone of chondrocytes in the growth plate of long bones and embryonic vertebrae. (3) Induction of a hypertrophic phenotype in progenitor sternal chondrocytes by treatment with ascorbate increases expression of Band 17. (4) Induction of hypertrophy in growth plate chondrocytes in short-term monolayer cultures correlates with a rapid but transient rise in Band 17 message. Our interpretation of these findings is that Band 17 expression is associated with the transition to hypertrophy, not maintenance of the hypertrophic phenotype. Molecular analysis of the 3' end of Band 17 cDNAs and genomic structure has shown that Band 17 is a single copy gene transcribed into four messages. Alternative splicing of these messages is predicted to result in two proteins that differ at the C-terminal by 131 amino acids. The longer protein contains a C-terminal consensus sequence that potentially targets this protein to the lumen of the endoplasmic reticulum. There is a Band 17 homologue in humans, suggesting conservation of Band 17 function in mammals. In summary, the pattern of expression and the predicted primary structure identify Band 17 as unique among all previously known chondrocyte genes.