In vitro bioassays using cell lines have been widely adopted for rapid screening of toxic chemicals. Different cell lines are often used for different toxicity tests according to their characteristics. For example, the Organization for Economic Co-operation and Development (OECD) used the RTgill-W1 cell line for assessing fish acute toxicity, whereas the H295R cell line with all enzymes regulating steroidogenesis is commonly used for assessing steroidogenic toxicity. However, since different cell lines are derived from specific species, organs and tissues, the responses interplayed with other organs or pathways thus cannot be reflected. Experimental results obtained from a specific cell line may not represent responses from the respective organ. To address whether the discrepancy between cell lines derived from the same organs is negligible, we have compared the differentially expressed genes (DEGs) of 2 female ovarian cell lines (SKOV3, COV434) and 2 male testicular cell lines (TM4, GC-1) treated with 9 endocrine disrupting chemicals (BPA, BP-3, BDE-47, DEHP, DHP, EE2, 4-NP, TCS, TDCPP). Our results showed that the number of DEGs differed drastically between cell lines from the same gender, and only a few dysregulated genes are common upon the same chemical treatment. Gene ontology enrichment analysis also suggested a diverse response in different cell lines. This raised questions on the reliability and representativeness of the present in vitro assays for toxicological assessment that draw conclusions based on data derived from one cell line only. Caution should be exercised in interpreting data obtained from in vitro bioassays.