Lutein may affect the bioefficacy of beta-carotene in humans. By using gas chromatography-combustion interfaced-isotope ratio mass spectrometry (GC-C-IRMS) and highly-enriched 13C-labeled tracers of beta-carotene and lutein, we compared the appearance of 13C-beta-carotene and its cleavage product, 13C-retinol, in plasma after subjects ingested 13C-beta-carotene with or without 13C-lutein. Women (n = 7) each ingested both a single dose of 1 mg perlabeled 13C-beta-carotene and a combined dose of 1 mg perlabeled 13C-beta-carotene and 3 mg perlabeled 13C-lutein in random order. Blood samples were collected at selected time intervals. Lutein, beta-carotene, and retinol were extracted from plasma and purified by using HPLC. Plasma retinol fractions were directly analyzed using GC-C-IRMS; lutein and Rcarotene were derivatized to thermally-stable derivatives which were analyzed using GC-C-IRMS. The appearance of 13C-beta-carotene in plasma was not different after the single or combined doses; nor was the appearance of 13C-retinol. The ratio of the 0--96 h area under the concentration vs. time curve (AUC) for 13C-retinol to the 0--528 h AUC for 13C-beta-carotene, an index for conversion of 13C-beta-carotene to 13C-retinol, was not different after the single or combined doses. Plasma beta-carotene concentration at baseline was negatively correlated with the 0--96 h AUC for 13C-retinol after the combined dose (r = -0.84, P = 0.02). Plasma lutein concentration at baseline was positively correlated with the 0--528 h AUC for 13C-lutein after the combined dose (r = 0.82, P = 0.02), and positively correlated with the 0--528 h AUC for 13C-beta-carotene after the single 13C-beta-carotene dose (r = 0.84, P = 0.02). The responses from the subjects to the doses were highly variable. The interindividual coefficient of variance (CV) of the 0--528 h AUC for 13C-beta-carotene was 59% after the single dose and 36% after the combined dose. The CV of the 0--96 h AUC for 13C-retinol was 64% after the single dose and 53% after the combined dose. The CV of the 0--528 h AUC for lutein was 44%. We conclude that, when ingested with beta-carotene in an amount and dose ratio typically found in dark green leafy vegetables, lutein does not affect the bioefficacy of beta-carotene.