Introduction: Non-invasive 13CO2-breath tests by using different 13C-labelled triglycerides are most widely used for evaluation of luminal lipase activity in pancreatic insufficient cystic fibrosis (CF)-subjects but lead to contradictory results. Aims: In the present study, the triglyceride oxidation was investigated in CF-subjects after administration of a 13C-labelled tripalmitate-trioleate mixture (TTM) and a synthetic mixed triglyceride (MTG) by comparing the 13CO2-breath exhalation. Methods: In a cross-over study, 6 male and 8 female CF-subjects (age: 7-30 years, body weight: 19.6 - 65.6 kg) were treated with Pangrol R (Berlin-Chemie, Berlin, Germany; dosage: 12,500 IU lipase/100 ml standard diet). The TTM ([1,1,1-13C3]glyceryl tripalmitate and [1,1,1-13C3]glyceryl trioleate, Cambridge Isotope Laboratories, Andover, U.S.A.; dosage: 4 mg/kg each) was administered as a single oral pulse at 8:00 am with the standard diet Fresubin R (Fresenius, Bad Homburg, Germany; dosage: 10 ml/kg). Later on, the same subjects were given the MTG (1,3-distearyl, 2[13C]octanoyl glycerol; dosage: 12.5 mg/kg) with the standard diet Nutri-Mix (Nutricia, Zoetemeer, Netherlands; dosage: 10 ml/kg). Breath samples were taken in 15- and 30-minute-intervals over 8 hours. The 13C-enrichment in CO2 of exhaled air was measured by continuous-flow isotope ratio mass spectrometry (Europa Scientific, Crewe, U.K.). Results: After administration of the TTM and the MTG the mean maximum 13CO2-enrichments were 3.62 and 7.52 delta over baseline, occurring at 7.0 and 3.5 hours, respectively. The corresponding percentage cumulative 13CO2-exhalations over the 8-hour time-span were 10.20% and 27.95%, respectively, and showed a statistically significant difference (P < 0.001). Discussion: After using the 13C-labelled TTM (containing palmitate and oleate as the predominant fatty acids of cows-milk fat) and the 13C-labelled MTG, the resulting 13CO2-exhalation reflects the triglyceride hydrolysis by luminal lipase as well as the absorption of glycerol, monoglycerides, free fatty acids and their subsequent oxidation. This suggests that the different metabolic fate of triglycerides and their respective fatty acids and the intestinal handling influences the results. It is concluded that the different cumulative 13CO2-exhalations were caused by chain length, degree of unsaturation of fatty acids and fat deposition but mainly by the rate limiting step of MTG-hydrolysis to stearate in the 1- and 3-positions. The resulting [13C]octanoyl monoglyceride or [13C]octanoate is then absorbed and rapidly metabolized to 13CO2. Conclusion: Non-invasive 13C-breath tests using different 13C-labelled triglycerides can be used for evaluation of pancreatic lipase activity, of different pancreatic enzyme preparations and for assessment of optimal lipase supplementation in CF-subjects.