AbstractHeparan sulfate (HS) is an unbranched chain of repetitive disaccharides, which specifically binds ligands when attached to the cell surface or secreted extracellularly. HS chains contain sulfated domains termed the HS fine structure, which gives HS specific binding affinities for extracellular ligands. HS 3‐O‐sulfotransferases (3‐OST) catalyze the transfer of sulfate groups to the 3‐O position of glucosamine residues of HS, a rare, but essential HS chain modification required for HS fine structure. We report here the first characterization and developmental expression analysis of the 3‐OST gene family in a vertebrate. There are eight 3‐OST genes in zebrafish: seven genes with homology to known 3‐OST genes in mouse and human, as well as a novel, 3‐OST‐7. A phylogenetic comparison of human, mouse, and zebrafish indicates the 3‐OST family can be subdivided into two distinct subgroups. We examined the mRNA expression patterns in several tissues/organs throughout early zebrafish development, including early cleavage stages, somites, brain, internal body organ primordial, and pectoral fin development. The 3‐OST gene family has both specifically expressed and ubiquitously expressed genes, suggesting in vivo functional differences exist between members of this family. Developmental Dynamics 235:3423–3431, 2006. © 2006 Wiley‐Liss, Inc.