The fractional extraction of unidirectional influx of several neutral amino acids (glutamine, leucine, alanine, tryptophan, and cycloleucine) into rat liver in vivo is studied with a tissue-sampling, single-injection technique. Liver uptake of 14C-amino acid is expressed as an index relative to the hepatic clearance of a 3H-labeled water (3HOH) internal reference. The maximal fractional extraction of 3HOH influx into liver, 0.85, and the rate constant of 3HOH exodus back to blood, 0.87 min-1, provide an estimate of portal blood flow, 0.93 ml min-1 g-1, in the barbiturate-anesthetized, laparotomized rat. Given the extraction data for the 3HOH reference, liver uptake indices for the five amino acids studied are converted into maximal fractional extractions of amino acid influx into liver: glutamine, 0.72 +/- 0.03; leucine, 0.56 +/- 0.02; alanine, 0.43 +/- 0.04; tryptophan, 0.40 +/- 0.03; cycloleucine, 0.25 +/- 0.01; and sucrose, 0.09 +/- 0.02. The influx of glutamine and cycloleucine is shown to be increased (35%) with 48 h of fasting. These data indicate that glutamine penetrates the liver cell membrane faster than any of the 19 amino acids studied thus far. The rate of unidirectional influx of glutamine and other amino acids into liver is estimated and reveals that the capacity of liver cells to transport amino acids is severalfold greater than that of other organs such as brain or muscle.