Objective: To discover a possible absorption and/or secretion of enterostatin into the circulating blood, as well as to compare the levels of circulating enterostatin after high-fat feeding and low-fat feeding. Research Methods and Procedures: Using a specific enzyme-linked immunosorbent assay, plasma enterostatin levels were determined after feeding a high-fat, a high-fat/sucrose, or a low-fat meal to Sprague-Dawley rats deprived of food overnight. Results: The enterostatin levels were increased by all diets; the response to the high-fat and the high-fat/-sucrose meals was greater in magnitude and duration than that to the low-fat meal. In addition, enterostatin levels correlated with the intake of dietary fat. Plasma enterostatin levels after high-fat feeding were found to be similar to those after intravenous administration of exogenous enterostatin known to inhibit high-fat food intake. Gel chromatography of pooled postprandial plasma extracts followed by high-performance liquid chromatography analysis showed that plasma enterostatin was identical to synthetic enterostatin. Affinity cross-linking of plasma proteins with (125)I-enterostatin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography, revealed a single band with a molecular weight of about 66 kDa, indicating the presence of a potential enterostatin-binding protein in plasma. Discussion: The measurements of plasma enterostatin may be a sensitive indicator for the measurement of fat intake.