Three substrates labeled with nonradioactive 13C have been employed to establish a trilogy of noninvasive breath tests to detect fat malabsorption in children and then to differentiate the etiology of the steatorrhea. Administration of 17 mg/kg of (13C)triolein Lipomul (The Upjohn Co., Kalamazoo, Mich.) resulted in a peak excretion rate of 13CO2 greater than 2.7% dose/h in 10 normal subjects (mean value 4.96 +/- 2.2% dose/h) whereas all 17 subjects with fat malabsorption were below this value (mean value, 0.75% +/- 0.63% dose/h); p less than 0.001). For the detection of fat malabsorption, the discriminative value of (13C)triolein was superior, 100% sensitive, and 89% specific, while the use of (13C)palmitic acid (17 mg/kg) or (13C)trioctanoin (7.5 mg/kg) alone yielded both false-positive and false-negative results. In 6 out of 6 cases, pancreatic insufficiency could be differentiated from mucosal disease (7 patients) or bile salt deficiency (4 patients) by the presence of abnormal triolein or trioctanoin breath tests, or both but normal palmitic acid breath tests. However, further differentiation of mucosal disorders from bile salt disorders could not be achieved using either a single- or a multiple-substrate breath test. The use of the single triolein breath test in children offers an attractive, sensitive alternative to conventional fecal fat measurements to establish the presence of steatorrhea, and when using 12C-lipid with multiple substrates, the tests are capable of providing additional insight into the mechanism of fat malabsorption.