In an attempt to define the basis for the microvascular changes observed in diabetic myocardium, a study was undertaken on the effect of elevated glucose on the synthesis by rat heart endothelial cells of the extracellular matrix components, types VI, IV and I collagen, as well as fibronectin. Confluent cultures of these cells, isolated by fluorescence-activated cell sorting after treatment with rhodamine-labelled acetylated low density lipoprotein, showed a three to fivefold enhancement in the synthesis of type VI collagen after exposure for 48 h to high glucose (20 to 30 mmol/l), as determined by immunoblot analysis. Increased production of type IV collagen and fibronectin was also observed, but the change was smaller and no effect on type I collagen was found. Measurement of mRNA levels by hybridization with cDNA probes indicated that 48-h exposure to high glucose significantly increased the level of transcripts for type VI and IV collagens but not for type I collagen. While glucose consumption by endothelial cells in high glucose doubled in the initial 24-h period, utilization returned to normal by 48 h, concomitant with a reduction in GLUT1 transcript levels, suggesting that signals for stimulation of collagen synthesis must be active during the initial period of exposure to elevated glucose levels.