Vascular endothelial growth factor (VEGF) is an extracellular matrix (ECM)-binding growth factor capable of driving neovascularization. VEGF can potentially be applied clinically via intramuscular (IM) injection to correct local ischemia associated with peripheral artery disease (PAD). As interactions with ECM elements and cognate receptors at the site of an IM injection define the local biology of VEGF and previous studies have only focused on systemic distribution measurements, we have established a method to monitor the local VEGF distribution and fate. Fluorescent-labeled VEGF was prepared that bound to ECM and activated a cognate receptor similarly to VEGF. Beginning by 2 h and becoming complete by 12 h following injection, fluorescence microscopy demonstrated the transition of labeled VEGF from an initial extensive interaction with ECM components to a focused labeling of vascular endothelial cells. Biochemical characterization verified the association of VEGF with ECM components and modification of endothelial cell function associated with vascular permeability changes known to accompany VEGF actions. Our data provide information concerning temporal and spatial VEGF fate and actions at the site of an IM injection that can help guide decisions regarding the identification of acceptable formulation strategies.