Assay of the activity of β-1,4-galactosyltransferase (β-1,4-GT) revealed that in addition to serum, milk, colostrum, amniotic and cerebrospinal fluids and malignant effusions, this enzyme is present also in tears and saliva. Molecular-sieve chromatography of human colostral whey and serum and subsequent assay of β-1,4-GT activity have shown that β-1,4-GT was present as a free enzyme (55 kDa) and associated with components of larger molar mass. The elution pattern did not change when the chromatography was carried out in a buffer devoid of, or enriched with, Mn2+, a cofactor of β-1,4-GT activity. However, the activity associated with the large molar mass components was absent when the chromatography was carried out in the presence of a chelating agent (EDTA). Analyses of the eluted material by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), and by immunodiffusion indicated that the major colostral component in β-1,4-GT activity-containing fractions was secretory IgA (S-IgA); in addition, the β-1,4-GT activity was detected in fractions that contained lactoferrin and α-lactalbumin. Interactions of β-1,4-GT with S-IgA and lactoferrin in colostrum were also demonstrated by the detection of radioactivity in precipitin lines obtained by immunoelectrophoresis and autoradiography of the colostral whey after it had been incubated with UDP-[3H]-galactose. Furthermore, radioactively labeled S-IgA and α-chain were detected when colostral whey incubated with UDP-[3H]-galactose was analyzed by SDS-PAGE under non-reducing and reducing conditions, respectively. In serum, the β-1,4-GT-binding components identified in fractions after molecular-sieve chromatography were IgG, IgA, IgM and transferrin. The binding of β-1,4-GT to immunoglobulins (Ig) was also demonstrated by assaying the β-1,4-GT activity associated with Sepharose-4B-immobilized Ig of various isotypes and molecular forms, which were incubated with colostral β-1,4-GT in the presence of Mn2+. β-1,4-GT measured by enzyme activity was bound to these Ig in order: polymeric IgA2 > monomeric IgA1 = polymeric IgA1 = secretory IgA = pentameric IgM > IgG. Immobilized component chains, namely α, μ and J chains, bound β-1,4-GT more effectively than native Ig. Incubation of the IgA1 myeloma protein with crude human colostral galactosyltransferase in the presence of UDP[3H]-galactose and Mn2+ resulted in galactosylation of both N- and O-linked carbohydrate side chains. This finding suggests that in addition to β-1,4-GT, β-1,3-GT, which is also present in human colostrum, may bind to IgA1 or other suitable substrates. The observed reactivity of some anti-SC antibodies with β-1,4-GT was due either to contamination of the immunogen, or to the sharing of common epitopes, which could be carbohydrates. © 1993.