We have investigated the effect of a glycosylphosphatidylinositol anchor on the distribution of the soluble lysosomal enzyme cathepsin D. Only 10% of the chimeric protein (CD-GPI) could be detected on the plasma membrane after transfection in CHO cells. Similarly to endogenous cathepsin D, intracellular CD-GPI was detected in vesicular structures, suggesting that CD-GPI is targeted to lysosomes. CD-GPI is present as three forms with M(r) 55, 50 and 37 kD which could correspond to the precursor, intermediate and mature forms of cathepsin D, respectively. CD-GPI was shown to be GPI anchored by differential extractability with Triton X-114 before and after phosphatidylinositol phospholipase C hydrolysis. Intracellular CD-GPI is mainly substituted with oligosaccharides containing uncovered mannose 6-phosphate residues whereas these residues are covered in the cell surface precursor form of CD-GPI. Ammonium chloride treatment reduces the lysosomal delivery of CD-GPI and increases the cell surface expression of its precursor form.