A new method for assaying the activity of the enzyme that catalyzes the formation of a cancer-associated glycolipid, paragloboside ( (\mathbf{n L c _ { 4 }}) Cer), from lactotriaosylceramide ( (\mathrm{Lc}{3} \mathrm{Cer}) ) and UDP-galactose has been developed that is based on a time-resolved fluoroimmunoassay (TRFIA) with a Europium (Eu)-chelate-labeled antibody. The substrate, (L{3} C e r), immobilized on a microtiter plate, was incubated with UDP-galactose, (\mathbf{M n C l}{2}), Triton CF-54, and the enzyme. The content of the incubation product, (\mathrm{nLc}{4} \mathrm{Cer}), was determined by the TRFIA with anti-nLe ({ }{4}) Cer monoclonal antibody (\mathrm{H}-11) as the first antibody and Eu-labeled anti-mouse IgM antibody as the second one. The lower limit of detection of (\mathrm{nLc}{4}) Cer was estimated to be (0.2 \mathrm{pmol}). This method was used to detect the galactosyltransferase activity in sera from patients with colorectal cancer or benign colorectal adenomas and from healthy subjects of a reference sample group. The reference interval was 0-0.25 pmol (/ 25 \mu \mathrm{l}) serum (/ 2) h. Activity was significantly greater in patients with colorectal cancer than in those with colorectal benign adenoma ((P<0.05)) and the subjects of the reference sample group ((P<0.01)). (c) 1994 Academic Press, Inc.