Absorption and metabolism of [C-13]9-cis-beta-carotene ([C-13]9c beta C) was studied in three subjects after a single oral dose. Subjects given 1.0 mg [C-13]beta-carotene (mean: 99.4% 9-cis-beta-carotene, 0.6% all-trans-beta-carotene; dose A) had substantial concentrations of [C-13]all-trans-beta-carotene ([C-13]tr beta C) and [C-13]all-trans retinol ([C-13]retinol) but very low concentrations of [C-13]cis-beta-carotene ([C-13]cis beta C) in saponified plasma 5 h after dosing, as determined by HPLC and isotope-ratio mass spectrometry. There was no evidence of appreciable absorption of [C-13]9-cis retinol. To determine the proportion of [C-13]tr beta C and [C-13]retinol derived from [C-13]9c beta C, a second set of studies in the same subjects was performed with the same isomeric composition except with C-13 labeling only in all-trans-beta-carotene (dose B). The results indicated that >95% of plasma [C-13]tr beta C and [C-13]retinol observed after dose A was derived from [C-13]9c beta C. The concentrations of [C-13]tr beta C observed, in excess of that derived from the trace amounts of [C-13]tr beta C in the dose, indicated that a significant proportion of the [C-13]9c beta C dose was isomerized to [C-13]tr beta C before entering the bloodstream. Although precise quantitative estimates of the extent of isomerization of 9-cis-beta-carotene could not be made, it is apparent that cis-trans isomerization of 9-cis-beta-carotene to all-trans-beta-carotene contributed to the near absence of postprandial plasma 9-cis-beta-carotene after its oral administration in humans. The observation of different ratios of beta-carotene to retinol between the two dosing protocols suggests that isomerization did not occur exclusively before uptake by the intestinal mucosa. These results indicate that isomerization of ingested 9-cis-beta-carotene before its secretion into the bloodstream limits the potential supply of 9-cis retinoids to tissues, and increases the vitamin A value of 9-cis-beta-carotene.