Analogs of the potent inhibitor of glucosylceramide(GlcCer) synthase, D-threo-l-phenyl-2-pahnitoylamino-3-pyrrolidino-l-propanol(P4), based on substitutions in the palmitoyl group were made by means of a stereo-selective synthetic method in order to elucidate the role of the hydrophobic portion in both the inhibitory action toward the enzyme and the biological effects. While P4 strongly inhibited GlcCer synthase with an IC50 of 0.5 ƒÊM in vitro, it also inhibited cell growth by 50% at the concentration of 7 ƒÊM. The shorter N-acyl chain analogs including decanoyl, octanoyl, and hexanoyl groups showed similar ICso values for GlcCer synthase(around 2 µM), but the hexanoyl analog exhibited only a slight inhibitory effect on cell growth, showing the dissociation between GlcCer deple tion and cell growth. Several compounds which exhibit similar hydrophobicity to the hexanoyl analog of P4 were subsequently designed. We found that D-threo-l-phenyl-2-benzyloxycarbonylamino-3-pyrrolidino-l-propanol(PBPP) was a most potent inhibitor, showing an IC5o of 0.3 ƒÊM. In cultured cells, PBPP was able to deplete glycosphingolip ids without affecting cell growth or the ceramide level.