To demonstrate that the lipid volume fraction in liver steatosis can be accurately estimated with in vivo hydrogen-1 magnetic resonance (MR) spectroscopy, the authors developed a calibration procedure based on in vitro MR spectroscopy of lipid extracts from steatotic liver specimens. The lipid volume fractions determined with the calibration procedure were compared with the results of histomorphometry and with calibrated computed tomographic (CT) data. The volume fraction of fat determined with MR spectroscopy was in good agreement with the CT results, whereas histomorphometry underestimated the amount of hepatic fat. The results indicate that determination of the fat volume fraction in steatotic liver can be achieved noninvasively with MR spectroscopy.