Hepatocyte growth factor/scatter factor (HGF/SF) is a paracrine growth factor which increases cellular motility and has also been implicated in tumor development and progression and in angiogenesis. Little is known about the metabolic alteration induced in cells following MetHGF/SF signal transduction. The hypothesis that HGF/ SF alters the energy metabolism of cancer cells was investigated in perfused DA3 murine mammary cancer cells by nuclear magnetic resonance (NMR) spectroscopy, oxygen and glucose consumption assays and confocal laser scanning microscopy (CLSM). ³¹P NMR demonstrated that HGF/SF induced remarkable alterations in phospholipid metabolites, enhanced the rate of glucose phosphorylation (P < .05). ¹³C NMR measurements, using [¹³C₁] -glucose-enriched medium, showed that HGS/SF reduced the steady state levels of glucose and elevated those of lactate (P < .05). In addition, HGF/SF treatment increased oxygen consumption from 0.58±0.02 to 0.71±0.03 ymol/hour per milligram protein (P < .05). However, it decreased CO₂ levels, attenuated pH decrease. The mechanisms of these unexpected effects were delineated by CLSM, using NAD(P)H fluorescence measurements, which showed that HGF/SF increased the oxidation of the mitochondrial NAD system. We propose that concomitant with induction of ruffling, HGF/SF enhances both the glycolytic and oxidative phosphorylation pathways of energy production.