Cobalamin (Cbl) utilization as an enzyme cofactor is dependent on its efficient transit through lysosomes to the cytosol and mitochondria. We have previously proposed that pathophysiological perturbations in lysosomal function may inhibit intracellular Cbl transport with consequences for down-stream metabolic pathways. In the current study we used both HT1080 fibroblasts and SH-SY5Y neurons to assess the impact that protease inhibitors, chloroquine and leupeptin, have on the distribution of [57Co] Cbl in lysosomes, mitochondria and cytosol. Under standard cell culture conditions the distribution of [57Co] Cbl in both neurons and fibroblasts was approximately 5% in lysosomes, 14% in mitochondria, and 81% in cytosol. Treatment of cells with either 25 mM chloroquine or 40 mM leupeptin for 48 h significantly increased the lysosomal [57Co] Cbl levels, by 4-fold in fibroblasts and 10-fold in neurons, and this was associated with reduced cytosolic and mitochondrial [57Co] Cbl concentrations. Based on western blotting of LAMP2 in fractions recovered from an OptiPrep density gradient, lysosomal Cbl trapping was associated with an expansion of the lysosomal compartment and an increase in a subpopulation of lysosomes with increased size and density. Moreover, the decreased mitochondrial Cbl that was associated with lysosomal Cbl trapping was correlated with decreased incorporation of [14C] propionate into cellular proteins / macromolecules, indicating an inhibition of Cbl-dependent methylmalonyl-coenzyme A mutase activity. These results add support to the idea that lysosomal dysfunction may significantly impact upon Cbl transport and utilization.