Receptor-binding of "high-uptake" forms of lysosomal enzymes to human diploid skin fibroblasts had been predicted from the Michaelis--Menten kinetics of uptake of these enzymes [e.g., Sando, G.N. & Neufeld, E.F. (1977) Cell 12, 619--627]. We have now demonstrated such binding directly by using a sensitive assay for the bound enzyme. Cells deficient in alpha-L-iduronidase were detached from plastic dishes by mild trypsinization, allowed to recover, and used in suspension. They were incubated with urinary alpha-L-iduronidase at 0 degrees C for 90 minutes and then washed by centrifugation through concentrated bovine serum albumin; the activity of the cell-associated enzyme was measured with 4-methylumbelliferyl alpha-L-iduronide as substrate. A Scatchard analysis showed 14,000 binding sites per cell and a Kd of 1 x 10(-9) M for high-uptake alpha-L-iduronidase; binding of the low-uptake form was barely detectable. Mannose 6-phosphate, a known competitive inhibitor of uptake, inhibited the binding competitively, with Ki = 1 x 10(-4) M. Unexpectedly, mannose 6-phosphate greatly accelerated the dissociation of bound enzyme. During uptake of alpha-L-iduronidase at 35 degrees C, the receptors were regenerated every few minutes, even in the absence of protein synthesis.