Background: The amounts of vitamin A that are metabolically derived from specific carotene-containing foods are largely unknown. Objective: We sought to develop an improved method for estimating the metabolic vitamin A potential of provitamin A carotenoids by using [H-2(4)]retinyl acetate (d(4)-RA) as an extrinsic reference standard. Design: Healthy subjects consumed a standardized test meal containing 6 mg beta -carotene as either raw carrot or spinach, either 20 or 1 g added fat, and 6.0 mu mol d(4)-RA. Concentrations of unlabeled (d(0)) retinyl esters (RE), labeled (d(4)) RE, and carotenoids in the plasma triacylglycerol-rich lipoprotein fraction (d<1.006 kg/L) were determined in serial blood samples with HPLC and gas chromatography-mass spectrometry. Baseline-corrected areas under the curve for d(0)-RE, d(4)-RE, and carotenoids were calculated, and the masses of absorbed do-retinol and carotenes were estimated assuming 80% absorption of the d(4)-RA reference dose. Results: In trials with ample (20 g) fat (n=6), 74% of the 6 mg beta -carotene ingested was taken up as beta -carotene plus RE with 0.3 +/-0.1 mg as retinol. Test meals without carotenes yielded no beta -carotene or d(0)-RE response and there was no effect of treatment (either fat amount or vegetable, n=6) on the mean d(4)-RE area under the curve. The lower-than-expected vitamin A yields were attributed to poor intestinal uptake rather than to low conversion of beta -carotene to RE. Conclusion: The triacylglycerol-rich lipoprotein and d(4)-RA method, which controls for variation in chylomicron kinetics in vivo and RE recovery during analysis, is useful for obtaining quantitative estimates of the vitamin A potential of single meals.