The influence of interferon-gamma (IFN-gamma), an activator of macrophages, was investigated on angiogenesis in vitro from rat aortic endothelial cells (EC). Subcultured EC were cultured in 0.15% type I collagen gel with 2% fetal bovine serum (FBS)-Dulbecco's modified Eagle's medium. Tube formation by EC began on the first day of culture and reached a plateau that lasted from the second to the eighth day. A peritoneal macrophage preparation was co-cultured on overlaying collagen gel containing EC. The macrophage preparation increased tube length from the second to the fourth day in a time-dependent manner. IFN-gamma (2.2 and 6.5 ng/ml) enhanced the effect of co-cultured macrophages from the fourth to the eighth day. The conditioned media derived from macrophages after 4 and 6 days of exposure to IFN-gamma (6.5 ng/ml) also showed significantly enhanced tube formation induced by macrophage-conditioned medium. However, IFN-gamma (6.5 ng/ml) did not influence the activity of the macrophage-conditioned medium. These results suggest that IFN-gamma enhances angiogenesis from EC of rat aorta by releasing an angiogenic factor from macrophages.