The effect of neuraminidase treatment on the lipid fluidity of the porcine intestinal brush-border membranes was studied using two fluorescence dyes, pyrene and 1,6-diphenyl-1,3,5-hexatriene. By treatment of the membranes with neuraminidase, the fluorescence parameters of pyrene-labeled membranes changed; i.e., a shift of thermal transition temperature, an increase in the fluorescence quenching rate for Tl+ and a decrease in the fluorescence lifetime. These results suggest that the environmental properties around the dye molecules in the membranes change sensitively upon neuraminidase treatment. Perturbation of the lipid domain in the membranes associated with neuraminidase treatment is also demonstrated by a stimulated solubilization of diphenylhexatriene molecules in the membrane lipids, an increased quenching efficiency with Tl+ and a decreased rotational correlation time of diphenylhexatriene-labeled membranes. Based on these results, we conclude that the lipid organization of the membranes is susceptible to neuraminidase treatment and that the membrane lipid fluidity increases by desialylation by the enzyme treatment.