The main goal of this study was to determine the temporal and spatial relationship of several components of the extracellular matrix (ECM) to coronary vascularization during prenatal and early postnatal development. Rat microvessels were visualized by immunolabeling for platelet endothelial cell adhesion molecule (PECAM-1), and by exposure to the lectin from Griffonia simplicifolia I. Coronary vasculogenesis, which first occurs in gestation day 13 (E13) hearts, was preceded by the deposition of fibronectin. The onset of laminin immunoreactivity in basement membranes coincided with tube formation and was followed by the appearance of collagen IV. Discontinuous collagen IV staining of basement membranes typified early tube formation but progressed to completely encircle capillaries. Sparse staining of collagen I and III was observed in prenatal hearts, but increased after birth. Staining for both molecules was limited mainly to the adventitia of vessels larger than capillaries, and as a component of septa and the epicardium. To determine the effects of loading conditions on key ECM molecules relating to neovascularization, avascular E12 rat hearts were grafted to the anterior eye chamber of adult hosts. In these hearts, which are hemodynamically unloaded, the appearance and distribution of ECM components were similar to hearts developing in utero. It was concluded that during heart development: (1) fibronectin may provide a primary scaffolding for the migration of primordial endothelial cells/angioblasts; (2) tube formation coincides with lamin deposition and is closely followed by the appearance of collagen IV; (3) collagens I and III are not related to tube formation in the prenatal heart; and (4) the relationship of the ECM to vessel formation is not notably altered in the absence of a ventricular load. Furthermore the early onset of PECAM-1 immunoreactivity suggests that it is a useful endothelial marker and may play a role in tube formation.