Cell culture systems provide an opportunity to evaluate the effects of carotenoids on molecular and cellular processes involved in proliferation and differentiation of prostate cancer cells. The stability and cellular uptake of beta-carotene (BC) by prostate cancer cells were investigated in vitro by use of various delivery methods and three human prostate adenocarcinoma cell lines: PC-3, DU 145, and LNCaP. Recovery of BC from the media (prepared from water-dispersible BC beadlets) significantly (p < 0.05) decreased after 12 hours in culture and continued to significantly decrease (p < 0.05) after 24, 48, 72, and 96 hours, an observation primarily attributed to BC degradation rather than isomerization, metabolism, or cellular uptake. The uptake of BC by prostate cancer cells was compared when delivered by tetrahydrofuran, BC-enriched bovine serum, water-dispersible BC beadlets, and artificial liposomes. Recovery of BC after three days in culture from enriched bovine serum medium was significantly (p < 0.05) greater than recovery from medium prepared by beadlets, tetrahydrofuran, or artificial liposomes. We conclude that BC is relatively unstable in vitro and that degradation products may contribute to biological responses. Furthermore, our studies indicate that enriched bovine serum provides a stable and physiological approach to carotenoid treatment of cells in culture.