The organophosphorus esters tris-(2-chloroethyl)-phosphate (TCEP) and tris-(2-chloropropyl)-phosphate (TCPP) have been widely used as flame retardants and fire preventing agents, e.g. in polyurethane foams. We investigated the cytotoxic, genotoxic, mutagenic, and estrogenic potentials of TCEP and TCPP, using different in vitro models. Cytotoxic effects were evaluated by neutral red uptake and genotoxicity with the alkaline single cell gel electrophoresis (Comet assay), both in V79 (hamster fibroblasts) cells. Mutagenicity was tested in the Ames assay with Salmonella typhimurium using the strains TA 97a, 98, 100, 102, 104, 1535, 1537, and 1538, with and without metabolic activation by S9-rat liver homogenate. Estrogenic or anti-estrogenic effects were examined with the recombinant yeast reporter gene assay, and in human endometrial cancer Ishikawa cells by induction of alkaline phosphatase. In V79 cells TCEP was weakly cytotoxic at concentrations above 10 μM in the presence of S9-rat liver homogenate whereas TCPP showed cytotoxicity above 1 mM in the presence of S9. Both substances did not induce DNA strand breaks in the alkaline version of the Comet assay neither without an external enzymatic metabolizing system, nor in the presence of S9-mix. Additionally, no mutagenic potential could be detected for TCEP and TCPP in eight Salmonella strains using concentrations up to 1 mM in the presence and absence of S9. Hormonal activity shown as induction of estrogenic or anti-estrogenic effects could not be detected in the two in vitro test systems.