Carotenoid bioavailability depends, amongst other factors, on the food matrix and on the type and extent of processing. To examine the effect of variously processed spinach products and of dietary fiber on serum carotenoid concentrations, subjects received, over a 3-wk period, a control diet (n = 10) or a control diet supplemented with carotenoids or one of four spinach products (n = 12 per group): whole leaf spinach with an almost intact food matrix, minced spinach with the matrix partially disrupted, enzymatically liquefied spinach in which the matrix was further disrupted and the liquefied spinach to which dietary fiber (10 g/kg wet weight) was added. Consumption of spinach significantly increased serum concentrations of all-trans-beta-carotene, cis-beta-carotene, (and consequently total beta-carotene), lutein, alpha-carotene and retinol and decreased the serum concentration of lycopene. Serum total beta-carotene responses (changes in serum concentrations from the start to the end of the intervention period) differed significantly between the whole leaf and liquefied spinach groups and between the minced and liquefied spinach groups. The lutein response did not differ among spinach groups. Addition of dietary fiber to the liquefied spinach had no effect on serum carotenoid responses. The relative bioavailability as compared to bioavailability of the carotenoid supplement for whole leaf, minced, liquefied and liquefied spinach plus added dietary fiber for beta-carotene was 5.1, 6.4, 9.5 and 9.3%, respectively, and for lutein 45, 52, 55 and 54%, respectively. We conclude that the bioavailability of lutein from spinach was higher than that of beta-carotene and that enzymatic disruption of the matrix (cell wall structure) enhanced the bioavailability of beta-carotene from whole leaf and minced spinach, but had no effect on lutein bioavailability.