We have characterized human plasma sialytransferase using a new fluorometric assay based on incorporation of a fluorescent NeuAc analogue into different acceptor glycoconjugates. This enables an exact characterization of acceptor specificity and kinetic properties. The data obtained indicate the presence of at least two distinct plasma sialytransferases: one specific for N-linked complex type glycan acceptors, and the other for GalNAc-residues on O-linked glycan acceptors. The first enzyme turned out to have very similar properties to a purified human liver sialytransferase, supporting an earlier hypothesis that liver is the main enzyme source. The new fluorometric assay presented here may be suitable for answering the question as to whether plasma sialytransferase is a useful diagnostic parameter in pathology.