To develop a protocol for measurement of liver fat using computed tomography (CT), we conducted a preliminary study with 118 men and 76 women to determine a readily identifiable vertebral landmark at which the CT image displayed both liver and spleen. Analysis of 5 landmarks revealed that the CT image obtained at the T12-L1 level simultaneously displayed the liver and spleen in 90% of the men and women. The T12-L1 protocol was cross-validated on a sample of 130 men and 113 women. In this sample we also assessed the regional characteristics of liver and spleen tissue attenuation at the T12-L1 level by subdividing each image into quartiles from anterior to posterior, each of which were further divided into medial and lateral regions. A similar analysis was performed on images located 6mm above and below T12-L1. The T12-L1 image displayed both liver and spleen in 92% (403/437) of the combined study sample. There was a significant (P < 0.005) stepwise increase in attenuation values (Hounsfield units (HU)) from the inferior to superior image. Although some significant (P < 0.05) differences were observed between the 8 regions by comparison to the whole liver or spleen, the average magnitude of the difference was <2.0 HU for liver and <3.5 HU for spleen. Acquisition of a single CT image at the T12-L1 level is a practical and reliable method for routine measurement of liver fat in research and clinical settings.