Purpose: The objective of this study was to determine the cytocompatibility of three different extracts of denture base resins and to compare the cytotoxic effect of these materials on a human oral epithelial KB cell line and primary human oral fibroblasts derived from buccal mucosa. Materials and Methods: Set specimens from a heat-cured resin, a self-cured resin, and a light-cured resin were eluted with culture medium for 1, 3, and 5 days. Cytotoxicity was judged using tetrazolium bromide reduction assay. Results: The eluates from self-cured, heat-cured, and light-cured denture base resins were cytotoxic to primary human buccal fibroblast cultures and KB cells. Self-cured resin was the most toxic denture base material among the chemicals tested in all cultures. The cytotoxicity decreased in the order of self-cured resin > heat-cured resin > light-cured resin for KB cells. The rank for buccal fibroblast cells was self-cured resin > heat-cured resin > light-cured resin. Conclusion: The influence of the cytotoxicity depended on the materials tested and the cell culture system used. The use of both permanent and primary cells is recommended for a better screening of the cytotoxic effects of denture base resins.