Avian β1,4 galactosyltransferase (GalTase) was purified from chicken serum, partially characterized, and compared to mammalian GalTase using antibody cross-reactivity, North-ern blot hybridization and amino acid sequence analysis. The enzyme was purified to apparent homogeneity by αlactalbumin(LA)-agarose affinity chromatography followed by preparative SDS-polyacrylamide gel electrophoresis, and identified as two proteins of apparent molecular masses of 39 and 46 kD. Chicken serum GalTase had a Km for UDPGal of 42 µM, for GlcNAc of 10 mM and had optimal activity in the presence of 10–20 mM MnCl2 Substrate and linkage specificity analyses indicated that the purified enzyme behaves as a traditional Gal β1,4 GlcNAc:GalTase, since: (i) the avian β1,4 GalTase bound to α-LA; (ii) terminal GlcNAc residues served as good acceptors for chicken serum GalTase; (iii) the enzyme was inhibited by high concentrations of GlcNAc; (iv) the galactosylated product was sensitive to β1,4-specific β-galactosidase. Finally, the disaccharide reaction product comigrated with authentic β1,4 N-acetyllactosamine standard. No other GalTase activities were detectable using a battery of defined glycoside substrates. Polyclonal antibodies raised against the two gel-purified GalTase proteins showed reactivity with avian GalTase by ELISA and immunoprecipitation assays. The antibodies also inhibited GalTase activity toward both high mol. wt and monosaccharide acceptor substrates. Despite similar kinetics and substrate specificity, the avian and mammalian GalTases showed little overall structural similarity, since polyclonal anti-avian GalTase IgG failed to react with mammalian GalTase purified from bovine milk, and conversely anti-bovine milk GalTase IgG did not react with the avian enzyme. Furthermore, in Northern blot analysis, no hybridization was detected when chicken embryo liver poly(A)+ RNA was probed with a mouse GalTase cDNA, even under conditions of reduced stringency. Amino acid sequence analysis identified three of five tryptic peptides that are homologous to the mammalian sequence within a putative substrate binding domain and the carboxy terminal domain of the enzyme. Their overall structural disparity leads us to believe that regions of homology between the avian and mammalian GalTases may represent active sites of the enzyme.