P>Aim To determine biocompatibility of three calcium phosphate cement (CPC) sealers, and to compare the cytotoxic response of human gingival fibroblasts (HGF) and one mouse fibroblast cell line (L929) to these materials. Methodology Monocalcium phosphate, calcium oxide and synthetic hydroxyapatite were combined with one of three aqueous solutions: modified polyacrylic acid, glass-ionomer liquid or 35% w/w polymethyl vinyl ether maleic acid to obtain Types I, IIa and III CPCs, respectively. Commercial Ca(OH)(2) sealer was used as a control. The materials were packed into Teflon molds (5.5 x 3 mm), and cellular function was assessed using MTT assay. The specimens were placed immediately in contact with cells, then evaluated at (24 h, 1 week, 2 week, 3 week, 4 week, 5 week). Results All materials showed significant cytotoxicity for both L929 and HGF cells at 24 h except for Type III. Type I was severely toxic initially, but improved significantly (P < 0.05) over the 5 week evaluation. Types II and Ca(OH)(2) were both cytotoxic over the 5 weeks. Type III CPC was equivalent to Teflon the entire time. The results showed the same rank of cytotoxicity in both cultures. The cytotoxic response decreased in the order of Type II > Ca(OH)(2) > Type I > Type III overtime. L929 cells were generally more sensitive than HGF cells to the calcium hydroxide-based sealer (Acroseal). Conclusion Types I and III have acceptable biologic properties for endodontic applications.