Blood glutamine is hydrolyzed by glutaminase; the resulting ammonia is dialyzed out and measured by use of a continuous-flow colorimetric microassay. Concentrations in the range of 10 to 1300 micromol of glutamine per liter can be determined in a 40-microliter sample. With slight modifications, higher concentrations, up to 5 mmol/liter, can be measured. Assay conditions and the qualities of the microtechnique were made optimal. Usual values obtained with this method for arterial and venous blood in man are, respectively, 443 +/- 140 and 466 +/- 142 micromol/liter (mean +/- 2 SD). Corresponding values for rats are 320 +/- 92 and 340 +/- 108 micromol/liter. Fasting causes a decreased blood glutamine.