Cytological appearance of fragile sites as non-staining gaps in metaphase chromosomes suggests an abnormality in chromatin structure. Studies of fragile sites at three levels of chromosome organization: (1) examining the ability of DNA derived from fragile sites to form nucleosomes-the basic structural element of chromosomes, (2) probing the arrangement of nucleosome arrays over fragile sites in fragile site-expressing cell lines, and (3) visualizing fragile sites in higher-order chromatin organization, reveal an unusual chromatin structure associated with fragile sites. This fragile site-associated chromatin structure might play an active role in DNA metabolic processes such as replication, transcription, repair and recombination, which are closely linked to the instability of fragile sites.