Total glutamine concentrations in commercial nutritional products have been determined by enzymatic hydrolysis followed by HPLC quantification of free glutamine and free pyroglutamic acid. Hydrolysis was accomplished by a published three-enzyme (Pronase, leucine aminopeptidase, prolidase), 20-h/37 degrees C digestion. Glutamine was determined as its FMOC derivative by reverse phase HPLC-fluorescence, and pyroglutamic acid was determined directly by organic acid HPLC-UV. Approximately 4.11% of the released glutamine is converted to pyroglutamic acid during the 20-h digestion. Experimental ratios of enzyme hydrolysis glutamine to acid hydrolysis glutamic acid + glutamine + pyroglutamic acid (GLX) indicate that the method recovers >90% of the protein-bound glutamine. The nutritional products with casein dominant intact protein systems typically deliver >9 g of glutamine/100 g of protein, or approximately 40 g of glutamine/100 g of GLX.