A series of 6- and 8-acylamino-4-methylumbelliferyl beta-D-galactopyranosides, beta-D-glucopyranosides, and alpha-L-fucopyranosides having various fatty acid residues were synthesized; 6-(9) and 8-hexadecanoylamino-4-methylumbelliferyl beta-D-galactopyranoside (10) were shown to be substrates for human galactocerebrosidase. Analogs of 9 with shorter acyl residues (octanoyl and butanoyl) were substrates for another type of beta-D-galactosidase, i.e., GM1-ganglioside-beta-D-galactosidase. The specificity of various beta-D-galactosidases for synthetic D-galactopyranosides, differing in the length and position of their acylamide residue, tested with enzyme preparations from patients with two types of glycolipidosis, Krabbe's disease (galactocerebrosidase deficiency) and GM1-beta-galactosidase deficiency), suggested that 9 is a specific substrate for galactocerebrosidase in biochemical tests for Krabbe's disease. Fluorogenic 6-octanoyl- and 6-hexadecanoyl-amino-4-methylumbelliferyl beta-D-glucopyranoside were much less readily hydrolyzed by both human and animal glucocerebrosidase than chromogenic 2-hexadecanoylamino-4-nitrophenyl beta-D-glucopyranoside. Comparison of the hydrolysis of 4-methylumbelliferyl alpha-L-fucopyranoside with that of 6-hexadecanoylamino-4-methylumbelliferyl alpha-L-fucopyranoside by multiple forms of human alpha-L-fucosidase showed that the enzyme is capable of hydrolyzing not only hydrophilic but also synthetic, lipid-like substrates.