Mucin-type O-glycosylation in Dictyostelium is initiated in the Golgi by a UDP-GlcNAc:polypeptide-Thr/Ser N-acetyl-alpha-glucosaminyltransferase (Dd-pp alpha GlcNAcT2) whose sequence is distantly related to the sequences of animal polypeptide-Thr/Ser N-acetyl-alpha-galactosaminyltransferases, such as murine Mm-pp alpha GalNAcT1. To evaluate the significance of this similarity, highly purified Dd-pp alpha GlcNAcT2 was assayed using synthetic peptides derived from known substrates. Dd-pp alpha GlcNAcT2 strongly prefers UDP-GlcNAc over UDP-GalNAc, preferentially modifies the central region of the peptide, and modifies Ser in addition to Thr residues. Initial velocity measurements performed over a matrix of UDP-GlcNAc donor and peptide acceptor concentrations indicate that the substrates bind to the enzyme in ordered fashion before the chemical conversion. Substrate inhibition exerted by a second peptide, and the pattern of product inhibition exerted by UDP, suggest that UDP-GlcNAc binds first and the peptide binds second, consistent with data reported for Mm-pp alpha GalNAcT1. Two selective competitive inhibitors of Mm-pp alpha GalNAcT1, retrieved from a screen of neutral-charge uridine derivatives, also inhibit Dd-pp alpha GlcNAcT1 competitively with only slightly less efficacy. Inhibition is specific for Dd-pp alpha GlcNAcT2 relative to two other Dictyostelium retaining glycosyltransferases. These data support a phylogenetic model in which the alpha GlcNAcT function in unicellular eukaryotes converted to an alpha GalNAcT function in the metazoan ortholog while conserving a similar reaction mechanism and active site architecture.