Apart from its ability to degrade extracellular matrix proteins, matrix metalloproteinase-2 (MMP-2) was recently revealed to have targets and actions within the cardiac myocyte. The localization of MMP-2 in caveolae of endothelial cells suggests that caveolin-1 (Cav-1) may play a role in regulating MMP-2. The caveolin scaffolding domain (CSD) of Cav-1 regulates several proteins including those involved with signaling cascades. Whether Cav-1 is responsible for regulating MMP-2 in the heart is unknown. Hearts from Cav-1(-/-) or Cav-1(+/+) mice were isolated and heart extracts or lipid raft enriched membrane fractions were prepared. MMP-2 activity in Cav-1(-/-) hearts was markedly enhanced when compared with Cav-1(+/+) hearts with no changes in MMP-2 protein levels between groups. In contrast, MMP-2 activity and protein level were greatly reduced in lipid raft enriched fractions of Cav-1(-/-) hearts. Purified CSD inhibited MMP-2 activity in a concentration-dependent manner as assessed using an in vitro degradation assay with a fluorogenic MMP-2 substrate (OmniMMP). These data suggest that Cav-1 plays a role in regulating MMP-2 activity. Cav-1 may thus be a novel mechanism to regulate MMP-2 activity in the heart.