Epidermal growth factor (EGF) or transforming growth factor‐α (TGF‐α) stimulates cell migration, proliferation and the formation of tube‐like structures of human microvascular endothelial cells in culture. Heparin‐binding EGF‐like growth factor (HB‐EGF), which shows 35% homology with EGF/ TGF‐α, is a member of the EGF family, and it is ubiquitous in many tissues and organs. We examined whether or not HB‐EGF induced angiogenic responses in human microvascular endothelial cells. HB‐EGF inhibited the binding of 125I‐EGF to the EGF receptor and induced autophosphorylation of the receptor on endothelial cells. Exogenous HB‐EGF induced the loss of more than 70% of the EGF receptor from the cell surface within 30 min, with similar kinetics to that of EGF. The level of c‐ fos mRNA markedly increased at 30 min in response to HB‐EGF as well as EGF. A gel shift assay demonstrated the activation of the transcription factor p91 by HB‐EGF and EGF. This factor directly interacts with the EGF receptor and mediates the activation of c‐ fos gene promoter. HB‐EGF enhanced the mRNA expression of tissue‐type plasminogen activator (t‐PA) and plasminogen activator inhlbitor‐1 (PAI‐1) mRNA. However, the enhancement of t‐PA and PAI‐1 by HB‐EGF was less than that by EGF. Heparitinase/chlorate, which digests the heparan sulfate proteoglycan of the endothelial cell surface, restored both t‐PA and PAI‐1 mRNA levels in response to HB‐EGF in a manner similar to that by EGF. HB‐EGF at 10 ng/ml developed tube‐like structures in type I collagen gel at similar levels to that of EGF at 10 ng/ml, suggesting that HB‐EGF is also a potent angiogenic factor in the model system for angiogenesis. The tubulogenesis activity of HB‐EGF is discussed in relation to the expression of the t‐PA and PAI‐1 genes.