AbstractAlthough a growing number of epidemiological studies indicate that dietaryβ‐carotene has anticarcinogenic activity, the mechanism(s) ofβ‐carotene protection remains to be definitively established. In this context,in vitrostudies ofβ‐carotene have been, and continue to be, valuable. We examined the following critical features in designing anin vitrosystem for studying the protective action ofβ‐carotene: 1) form ofβ‐carotene used for cellular uptake, 2) cellular metabolism ofβ‐carotene, and 3) subcellular distribution ofβ‐carotene. It was determined thatβ‐carotene added to medium in a water‐dispersible formulation is readily taken up by BALB/c 3T3 cells and is located predominantly in cellular membranes. Cellular uptake ofβ‐carotene added to medium in an organic solvent is greatly reduced. It was also found that intracellular retinol increased significantly after a three‐day exposure of BALB/c 3T3 cells to media containingβ‐carotene. This result suggests that the ability to metabolizeβ‐carotene to retinoids is not limited to cells of intestinal origin. The results and methodology described here will be useful in the rational design ofin vitroassays for elucidating the mechanism(s) ofβ‐carotene protective effects at the cellular level.