3-O-Sulphates are the rarest substituent of heparan sulphate and are therefore ideally suited to the selective regulation of biological activities. Individual isoforms of heparan sulphate D-glucosaminyl 3-O-sulphotransferase (3-OST) exhibit sequence-specific action, which creates heparan sulphate structures with distinct biological functions. For example, 3-OST-1 preferentially generates binding sites for anti-thrombin, whereas 3-OST-3 isoforms create binding sites for the gD envelope protein of herpes simplex virus 1 (HSV-1), which enables viral entry. 3-OST enzymes comprise a presumptive sulphotransferase domain and a divergent N-terminal region. To localize determinants of sequence specificity, we conducted domain swaps between cDNA species. The N-terminal region of 3-OST-1 was fused with the sulphotransferase domain of 3-OST-3(A) to generate N1-ST3(A). Similarly, the N-terminal region of 3-OST-3(A) was fused to the sulphotransferase domain of 3-OST-1 to generate N3(A)-ST1. Wild-type and chimaeric enzymes were transiently expressed in COS-7 cells and extracts were analysed for selective generation of binding sites for anti-thrombin. 3-OST-1 was 270-fold more efficient at forming anti-thrombin-binding sites than 3-OST-3(A), indicating its significantly greater selectivity for substrates that can be 3-O-sulphated to yield such sites. N3(A)-ST1 was as active as 3-OST-1, whereas the activity of N1-ST3(A) was as low as that of 3-OST-3(A). Analysis of Chinese hamster ovary cell transfectants revealed that only 3-OST-3(A) and N1-ST3(A) generated gD-binding sites and conveyed susceptibility to infection by HSV-1. Thus sequence-specific properties of 3-OSTs are defined by a self-contained sulphotransferase domain and are not directly influenced by the divergent N-terminal region.