Glycosaminoglycans (GAGs) from breast cyst fluid (BCF) of gross cysts, subdivided into apocrine and flattened, directly collected from 27 gross-cystic-breast-disease (GCBD)-affected women were analysed. Heparan sulfate, not further investigated, and chondroitin sulfate were identified. This last polysaccharide, in a content of 25–27 μg ml−1 BCF and having a high molecular mass (˜20 000–22 000), was found rich in glucuronic acid (˜96%–98%) and mainly sulfated in position 4 of the N-acetyl-galactosamine (˜60%–64%). Moreover, the presence of ˜19%–24% of uncommon 4,6-O-disulfated disaccharides CS-E inside the polysaccharide chains with a high charge density of ˜1.15–1.20 was determined. No substantial differences between apocrine and flattened cysts were observed. The current study describes the first effort to examine the yield and distribution of complex macromolecules like GAGs in BCF, and the understanding of their structure may help explain some functions associated with physiological and pathological conditions. Copyright © 2013 John Wiley & Sons, Ltd.