TPST1 is a human tyrosylprotein sulfotransferase that uses 3'phosphoadenosine-5'phosphosulfate (PAPS) to transfer the sulfate moiety to proteins predominantly designated for secretion. To achieve a general understanding of the cellular role of human tyrosine-directed sulfotransferases, we investigated targeting, structure and posttranslational modification of TPST1. Golgi localisation of the enzyme in COS-7 and HeLa cells was visualised by fluorescence imaging techniques. PNGase treatment and mutational studies determined that TPST1 bears N-linked glycosyl residues exclusively at position Asn60 and Asn262. By alanine mutation of these asparagine residues, we could determine that the N-linked oligosaccharides do not have an influence on Golgi retention of TPST1. In concert with N and C-terminal flanking residues, the transmembrane domain of TPST1 was determined to act in targeting and retention of the enzyme to the trans-Golgi compartment. This domain exhibits a pronounced secondary structure in a lipid environment. Further in vivo FRET studies using the transmembrane domain suggest that the human tyrosylprotein sulfotransferase may be functional as homodimer/oligomer in the trans-Golgi compartment.