The possibility of using exfoliated colonic epithelial cells for assessing the bioavailability of β-carotene was examined. Analysis of exfoliated colonic epithelial cells showed the presence of β-carotene and vitamin A. The β-carotene content was significantly lower in cells from stool samples of subjects on a β-carotene-poor diet than those receiving a single dose of a β-carotene supplement. Colonic epithelial cells isolated from stool samples collected daily during a wash-out period while the subjects were on a β-carotene-poor diet showed a steady decrease in β-carotene content, reaching the lowest value on day 7. Kinetic analysis showed that a single dose of a β-carotene supplement in the form of spirulina (Spirulina platensis) or agathi (Sesbania grandiflora) after the wash-out period caused an increase in the β-carotene content after a lag period of 5–7 d, but the vitamin A levels during these periods were not significantly affected. Analysis of plasma β-carotene concentration also showed similar changes, which correlated with those of exfoliated colonic cells. A relationship between the β-carotene content of the diet and that of the colonic epithelial cells suggests that analysis of the β-carotene content in exfoliated human colonic epithelial cells is a useful non-invasive method to assess the bioavailability of provitamin A β-carotene.