Although the liver is known to extract amino acids and organic anions by well-characterized transport systems, the factors that regulate the hepatic uptake of small, circulating peptides are poorly understood. We previously reported that cholecystokinin octapeptide, a biologically active form of cholecystokinin, is efficiently cleared by the liver and that uptake depends on its carboxyl-terminal tetrapeptide (Trp-Met-Asp-PheNH2). Here we further define the physicochemical determinants for hepatic clearance of cholecystokinin. A series of 13 tetrapeptides, including eight analogs of the carboxyl-terminal tetrapeptide of cholecystokinin-8 with different charges, hydrophobicity and aminoacid sequences, were prepared by solid-phase synthesis, purified by high-performance liquid chromatography and characterized by amino-acid analysis and mass spectrometry. Radioiodination was performed by oxidative or nonoxidative techniques. Hydrophobicity of individual radiolabeled peptides was calculated using published hydrophobicity data or measured directly by determining their partition between octanol and aqueous triethylammonium acetate. First-pass hepatic extraction of radiolabeled peptides was determined with a nonrecirculating, isolated, perfused rat liver model. First-pass hepatic extraction of injected, labeled peptides varied from 4% to 86% and correlated significantly (r = 0.85; p < 0.0002) with hydrophobicity. Hydrophobic peptides with positive, neutral or negative charges were avidly extracted (30% to 86%) by the liver; first-pass clearance of hydrophobic peptides with similar charges varied with amino-acid sequence. In contrast, the first-pass hepatic extraction of positively or negatively charged hydrophilic tetrapeptides was negligible (<10%). These results suggest that hydrophobicity and amino-acid sequence–but not anionic or cationic nature–are the major determinants of hepatic extraction of cholecystokinin, and perhaps other small, circulating peptides. (HEPATOLOGY 1990;12:76–82).