Endothelial cells (ECs) display phenotypic heterogeneity. Endothelial cell heterogeneity is mediated, at least in part, by site-specific and time-dependent differences in gene transcription. The goal of this review is to provide a conceptual framework for approaching EC gene regulation in the adult vasculature. We summarize data from cell culture studies that provide insight into the transcription factors involved in mediating gene expression in ECs. Next, we review the results of in vivo studies relating to gene regulation in the intact endothelium. Finally, we draw on both the in vitro and in vivo results to propose a model of gene regulation that emphasizes the importance of the extracellular environment in controlling site- and time-specific vascular gene expression.