Breast cancer cells oversecrete the lysosomal peptidase cathepsin D as a pro-enzyme. In this study, we assessed the effect of media conditioned by MRC-5 fibroblasts or MCF-7/6 breast cancer cells on cathepsin D (CD) production and secretion by these two cell types. We also considered the influence of estrogens and matrix components (type I or type IV collagen, or Matrigel) on the expression and activity of CD produced by breast cancer cells of different invasive potentials (MCF-7/AZ, MCF-7/6, MDA-MB-231). In our system, fibroblasts conditioned medium does not significantly affect CD levels produced and secreted by the MCF-7/6 cells. However, we found that fibroblasts are able to capture the pro-CD secreted by these tumor cells by a mannose 6-phosphate-dependent process. We also found a positive correlation between the proportion of extracellular CD levels and the invasive potentials of the tumor cell types considered. If estrogens are able to upregulate CD production and secretion by receptor-positive cells, it is not the case of extracellular matrix components. On the other hand, our results indicate that matrix components are able to influence the distribution of the different CD forms in and out of the cells. Our data suggest that tumor fibroblasts, by enhancing their intracellular CD levels, could assist cancer cells in the digestion of extracellular matrix during the invasion of tissues.