Transcription of the type II collagen gene (Col2a1) is regulated by multiple cis-acting sites. The enhancer element, which is located in the first intron, is necessary for high-level and cartilage-specific expression of Col2a1. A mouse limb bud cDNA expression library was screened by the Saccharomyces cerevisiae one-hybrid screening method to identify protein factors bound to the enhancer. A zinc finger protein, alphaA-crystallin binding protein 1 (CRYBP1), which had been reported to bind to the mouse alphaA-crystallin gene promoter, was isolated. We herein demonstrate that CRYBP1 is involved in the negative regulation of Col2a1 enhancer activity. CRYBP1 mRNA expression was downregulated during chondrocyte differentiation in vitro. In situ hybridization analysis of developing mouse cartilage showed that CRYBP1 mRNA was also downregulated during mesenchymal condensation and that CRYBP1 mRNA was highly expressed by hypertrophic chondrocytes, but at very low levels by resting and proliferating chondrocytes. Expression of recombinant CRYBP1 in a transfected rat chondrosarcoma cell line inhibited Col2a1 enhancer activity. Electrophoretic mobility shift assays showed that CRYBP1 bound a specific sequence within the Col2a1 enhancer and inhibited the binding of Sox9, an activator for Col2a1, to the enhancer. Cotransfection of CRYBP1 with Sox9 into BALB/c 3T3 cells inhibited activation of the Col2a1 enhancer by Sox9. These results suggest a novel mechanism that negatively regulates cartilage-specific expression of Col2a1.