It has been proposed that in cultured fibroblasts the final packaging of enzymes in lysosomes requires excretion followed by pinocytosis by neighboring cells via a carbohydrate-specific receptor mechanism. It has also been proposed that the abnormally high activity of lysosomal enzymes in the medium of cultured fibroblasts from patients with I-cell disease (mucolipidosis II) results from an altered carbohydrate recognition residue on the enzymes which prevents reuptake into the cells. With beta-hexosaminidase as a marker, and competitive inhibition of uptake by 2 mM mannose-6-phosphate, we have determined that only 12% of the total (intra- and extracellular) beta-hexosaminidase in normal fibroblasts is channeled through the excretion-reuptake route. After 9 d of exposure to mannose-6-phosphate, normal fibroblast cultures accumulated in the medium only a fraction of the enzyme excreted by I-cell disease fibroblasts in the same period. Furthermore, this minimal loss of enzyme to the medium did not result in a decrease of intracellular enzyme activity. Finally, if the defect in I-cell disease were only because of an impairment of a reuptake mechanism that involves only 12% of the total enzyme, then 88% of the newly synthesized enzyme should be retained by I-cell fibroblasts, resulting in intracellular activity three to nine times higher than that which is observed. These data are consistent with our previous proposal that excessive lysosomal enzyme activity in the medium of I-cell disease fibroblasts results from preferential exocytosis.