The kinetics of exposure of endocytosed material to two lysosomal enzymes were determined for a number of cultured cell lines using fluorogenic substrates. Hydrolysis of endocytosed substrates for cathepsin B and acid phosphatase was observed to begin within 3-10 min of substrate addition and to proceed linearly for up to 60 min thereafter. Hydrolysis of the cathepsin B substrate was not affected by inhibition of protein synthesis with cycloheximide, indicating that the enzymes present in early endosomes are not exclusively newly synthesized. As had been observed previously for a cathepsin B substrate (Roederer, M., Bowser, R., and Murphy, R. F., J. Cell. Physiol., 131:200-209, 1987), hydrolysis of the acid phosphatase substrate was not blocked at temperatures below 20 degrees C. The results suggest that the endosome is the primary site of initial exposure of endocytosed material to hydrolytic enzymes.