Endothelial monocyte-activating polypeptide-II (EMAP II) is an antiangiogenic factor for rapidly growing endothelial cells that is released from tumor cells under physiological stress such as hypoxia. We have previously shown that the interaction between EMAP II and the alpha-subunit of ATP synthase, alpha-ATP synthase, can play a regulatory function in the growth of endothelial cells. In the current study, we found that EMAP II-alpha-ATP synthase interaction could be inhibited by excess heparin, whereas the interaction could be enhanced by a low concentration of heparin. Both EMAP II and alpha-ATP synthase could specifically interact with heparin, and this interaction was increased under acidic conditions. In addition, EMAP II and alpha-ATP synthase were found to contain the heparin binding motifs determined by analysis using site-directed mutant forms. In endothelial cells, binding of EMAP II to cells was dramatically enhanced, and alpha-ATP synthase could associate with heparan sulfate at acidic pH. The inhibitory effect of EMAP II on the growth of cultured endothelial cells was also significantly enhanced at acidic pH. Analysis using mutant EMAP II proteins demonstrated that heparan sulfate was essential for the enhanced binding and EMAP II function to endothelial cells at acidic pH. Furthermore, the enhanced inhibitory effects of EMAP II could be abrogated by excess heparin or heparinase treatment. In the endothelial cell, heparan sulfate may regulate the function of EMAP II released from the tumor cell in hypoxic condition.