Matrix metalloproteinase-type 2 (MMP-2) degrades extracellular matrix, mediates cell migration and tissue remodeling, and is implicated in mediating neural crest (NC) and cardiac development. However, there is little information regarding the expression and distribution of MMP-2 during cardiogenesis and NC morphogenesis. To elucidate the role of MMP-2, we performed a comprehensive study on the temporal and spatial distribution of MMP-2 mRNA and protein during critical stages of early avian NC and cardiac development. We found that ectodermally derived NC cells did not express MMP-2 mRNA during their initial formation and early emigration but encountered MMP-2 protein in basement membranes deposited by mesodermal cells. While NC cells did not synthesize MMP-2 mRNA early in migration, MMP-2 expression was seen in NC cells within the cranial paraxial and pharyngeal arch mesenchyme at later stages but was never detected in NC-derived neural structures. This suggested NC MMP-2 expression was temporally and spatially dependent on tissue interactions or differed within the various NC subpopulations. MMP-2 was first expressed within cardiogenic splanchnic mesoderm before and during the formation of the early heart tube, at sites of active pharyngeal arch and cardiac remodeling, and during cardiac cushion cell migration. Collectively, these results support the postulate that MMP-2 has an important functional role in early cardiogenesis, NC cell and cardiac cushion migration, and remodeling of the pharyngeal arches and cardiac heart tube.