A high galactosyltransferase activity with lactotriaosylceramide as acceptor has been found in human serums. The reaction requires UDP-galactose, Mn2+, and Triton X-100. It has a pH optimum of 6.0 and a Km for lactotriaosylceramide of 0.66 mM. On the basis of methylation analysis and susceptibility towards beta-galactosidase the reaction product has been identified as neolactotetraosylceramide. Lactotetraosylceramide formation was not observed. Evidence is presented that the serum enzymic activity can be attributed to N-acetyllactosamine synthase (EC 2.4.1.90). The beta 1 leads to 4-galactosyltransferase activities with lactotriaosylceramide as acceptor have been determined in different human sera. The activity is independent of ABO, p and Rh blood-group status. The enzyme is within a normal range in serums of cancer patients.