Thrombin (THR), activated as part of the clotting cascade, dilates conduit arteries through an endothelial pertussis toxin (PTX)-sensitive G-protein receptor and releases nitric oxide (NO). THR also acts on downstream microvessels. Therefore, we examined whether THR dilates human coronary arterioles (HCA). HCA from right atrial appendages were constricted by 30-50% with endothelin-1. Dilation to THR (10^-4 to 1 U/ml) was assessed before and after inhibitors with videomicroscopy. There was no tachyphylaxis to THR dilation (max dilation = 87.0%, ED50 = 1.49x10^-2). Dilation to THR was abolished with either hirudin or denudation but was not affected by PTX. Neither L-NAME (n=7), INDO (n=9), ODQ (n=6), TEA (n=5) nor IBTX (n=4), reduced dilation to THR. However, KCl (max dilation=89±5vs.20±10%,p<0.05,n=7), TBA (max dilation=79±7vs.21±4%,p<0.05,n=5), and CTX (max dilation=89±4vs.10±2%,p<0.05,n=4) attenuated dilation to THR. In contrast to animal models, THR-induced dilation in human arterioles is independent of G_i-protein activation or NO release. However, THR dilation is endothelial dependent, is maintained on consecutive applications, and involves activation of potassium channels. We speculate that an endothelium derived hyperpolarizing factor contributes to thrombin-induced dilation in HCA.