We established distinctive monolayer and organotypic cell culture techniques to assess possible differences in cross-talk and spatial and structural organization of oral cancer cells compared with normal oral cells and also to evaluate possible differential responses of the cells to carotenoids. In monolayers, we investigated the effect of lycopene on the proliferation of an established oral cancer cell line, KB-1, and compared it with a primary cell line obtained from normal oral mucosa. Lycopene exerted a significant inhibitory effect on KB-1 cell proliferation inducing a dose-dependent downregulation of proliferating cell nuclear antigen (PCNA) associated with upregulation of connexin-43 (Cx-43) expression, whereas in the normal oral mucosal cells lycopene did not affect either PCNA expression, which was very low, or the expression of Cx-43, which was basically very high. Lycopene significantly inhibited the formation of colonies induced by the carcinogen 3-methylcholanthrene (MCA) on normal oral cells and almost completely abrogated the hyperplastic effect induced by MCA. KB-1 cells and normal oral epithelial cells in the organotypic cell culture method differed in their stratification and interrellular adhesion patterns as well as in the expression profile of cytokeratins, vimentin, and Cx-43. Lycopene induced Cx-43 expression in KB-1 cells grown by the organotypic raft method, similar to KB-1 cells grown as monolayers. We conclude that lycopene is a promising chemopreventive, pro-differentiating, and anticarcinogenic agent. No adverse effects of lycopene were detected in normal cells cultured in either monolayer or organotypic systems.