AbstractMatrix metalloproteinase‐2 (MMP‐2) is best understood for its biological actions outside the cell. However, MMP‐2 also localizes to intracellular compartments and the cytosol where it has several substrates, including troponin I (TnI). Despite a growing list of cytosolic substrates, we currently do not know the mechanism(s) that give rise to the equilibrium between intracellular and secreted MMP‐2 moieties. Therefore, we explored how cells achieve the unique distribution of this protease. Our data show that endogenous MMP‐2 targets inefficiently to the endoplasmic reticulum (ER) and shows significant amounts in the cytosol. Transfection of canonical MMP‐2 essentially reproduces this targeting pattern, suggesting it is the quality of the MMP‐2 signal sequence that predominantly determines MMP‐2 targeting. However, we also found that human cardiomyocytes express an MMP‐2 splice variant which entirely lacks the signal sequence. Like the fraction of ER‐excluded, full‐length MMP‐2, this variant MMP‐2 is restricted to the cytosol and specifically enhances TnI cleavage upon hypoxia‐reoxygenation injury in cardiomyocytes. Together, our findings describe for the first time a set of mechanisms that cells utilize to equilibrate MMP‐2 both in the extracellular milieu and intracellular, cytosolic locations. Our results also suggest approaches to specifically investigate the overlooked intracellular biology of MMP‐2. J. Cell. Physiol. 227: 3397–3404, 2012. © 2011 Wiley Periodicals, Inc.