Sialic acids are located at the terminal branches of the cell glycocalyx and secreted glycan molecules. O-Acetylation is an important modification of the sialic acids, however very few studies have demonstrated the in situ distribution of the O-Acetylated sialic acids. Here the distribution of glycoprotein bound 4-O-Acetylated sialic acids (4-O-Ac sias) in vertebrates was determined using a novel virus histochemistry assay. The 4-O-Ac sias were found in the circulatory system, i.e. on the surface of endothelial cells and RBCs, of several vertebrate species, though most frequently in the cartilaginous fish (class Chondrichthyes) and the bony fish (class Osteichthyes). The O-Acetylated sialic acid was detected in 64 % of the examined fish species. Even though the sialic acid was found less commonly in higher vertebrates, it was found at the same location in the positive species. The general significance of this endothelial labelling pattern distribution is discussed. The seemingly conserved local position through the evolution of the vertebrates, suggests an evolutionary advantage of this sialic acid modification.