Growing evidence that high glucose may be a causative agent of the thickened vascular basement membranes that characterize diabetic microangiopathy prompted this investigation of the underlying mechanisms. When exposed to 30 mM glucose, 70% of 52 primary cultures of human endothelial cells, each derived from a single umbilical vein, showed increased levels of fibronectin (median 181% of control, range 104–549%) and collagen IV mRNA (175% of control, range 101–807%). The response of the two transcripts to high glucose was concordant in 77% of the 52 cultures studied (P = 0.01), required 5 days of exposure, and was accompanied by proportionally increased synthesis of the respective protein. Laminin B1 expression was also upregulated by high glucose, concordantly with that of fibronectin and collagen IV. Increased fibronectin and collagen IV mRNA levels resulted from increased gene transcription (median 183 and 236% of control, respectively) without evidence of translational regulation, were not triggered by hypertonicity or signals originating from the matrix, and were also induced by hexoses with limited (D-galactose) or no (L-glucose) access to metabolic pathways but capable of inducing nonenzymatic glycosylation. There was no amplification of the overexpressed genes. Thus, high glucose upregulates in a coordinated fashion the transcription of genes coding for basement membrane components through effects exerted intracellularly or at the cell-matrix boundary and modulated by individual characteristics of the target cells.