The primary structure of galectin-3, a approximately 30 kDa galactoside-binding protein (aka CBP-35, mL-34, hL-31, L-29, Mac-2, and epsilon BP), reveals two structural domains: an amino-terminal domain consists of a Pro-Gly-rich motif, and a globular carboxyl-terminal domain containing a carbohydrate-binding site. In this study, we report that the amino-terminal domain of galectin-3 contains a cleavage site for two members of the matrix metalloproteinase family of enzymes: the 72 kDa (gelatinase A, MMP-2) and the 92 kDa (gelatinase B, MMP-9) proteinases. The major cleavage site for the gelatinases in galectin-3 is at the Ala62-Tyr63 bond, and its hydrolysis by these enzymes was inhibited by TIMP-2. Cell-surface expression of galectin-3 was reduced following treatment of viable T47D human breast carcinoma cells with gelatinase A. These results suggest that galectin-3 may be a substrate for gelatinases and that its degradation may play a role in modulating the biological activities of galectin-3.