A UDP-N-acetylgalactosamine:globotriaosylceramide beta-3-N-acetylgalactosaminyltransferase which catalyzes the conversion of human blood group Pk antigen into P antigen has been purified over 18,000-fold in 4% yield from a Triton X-100 extract of canine spleen microsomes by affinity chromatography on UDP-hexanolamine-Sepharose and globotriaosylceramide acid-Sepharose. The purified enzyme migrates as two major bands with apparent molecular weights of 64,000 and 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single band, with enzyme activity, was observed in nondenaturing acrylamide gels containing Triton X-100. Mn2+ was required for activity, and the pH optimum was 6.9. Km values for UDP-GalNAc and globotriaosylceramide were 14 and 2.5 microM, respectively. Studies on substrate specificities indicate that the preferred substrates have the general structure Gal alpha 1-4Gal-OR in which the nature of the R moiety has relatively little effect on activity. An antibody against the purified enzyme eliminated the activity of the enzyme, but did not neutralize the alpha-3-N-acetylgalactosaminyltransferase involved in the biosynthesis of Forssman glycolipid.