Leukocyte adhesion deficiency type II (LAD II) is a rare genetic disease characterized by severe immunodeficiency which is related to defective expression in leukocytes of sialyl-Lewis X (SLeX), a fucosylated ligand for endothelial selectins. The molecular basis of LAD II is still unknown, but has been tentatively localized in the de novo pathway of GDP-L-fucose biosynthesis from GDP-D-mannose. Here, we demonstrate that in cell lysates from a LAD II patient, GDP-D-mannose-4,6-dehydratase (GMD), the first of the two enzymes of the pathway has a defective activity compared to control subjects. GMD in cell lysates from both parents showed intermediate activity levels. Cloning of GMD from patient and control lymphocytes ruled out any mutation affecting the amino acid GMD sequence and the purified recombinant proteins from both controls and the patient showed identical specific activities. Since the levels of immunoreactive GMD in cell lysates were comparable in the patient and in controls, the biochemical deficiency of intracellular GMD activity in LAD II seems to be due to mutation(s) affecting some still unidentified GMD-regulating protein.