AbstractEmbryonal carcinoma (EC) cells are recognized as an excellent model system for studying the early stages of mammalian development. Many studies performed with EC cells involve transient transfection with promoter/reporter gene constructs and/or mammalian expression vectors. One of the limitations of working with EC cells is their inability to be transfected at high efficiency. In most cases, EC cells are transfected using the calcium phosphate method. The objective of this study was to identify protocols and culture conditions that significantly increase the transfection efficiency of EC cells. F9 EC cells were used for this purpose, because they are the EC cell line studied most commonly. We show that the transfection efficiency of F9 EC cells using the calcium phosphate method is less than 5%; whereas, their transfection efficiency can be improved ∼15‐fold using optimized culture conditions and liposome‐based transfection reagents. Specifically, we demonstrate that more than 50% of F9 EC cells can be transfected using LipofectAMINE 2000. In addition to higher levels of transfection, there is much less plate‐to‐plate variation with liposome‐based reagents as compared to transfection with calcium phosphate. Interestingly, transfection efficiency using these reagents was found to be inversely related to cell density. This contrasts sharply with the recommendation that transfection with LipofectAMINE 2000 or LipofectAMINE in conjunction with the PLUS reagent be performed at high cell densities. Given the improvements in transfection efficiency reported here, it will now be possible to perform studies with F9 EC cells that require transfection at significantly higher levels than that achieved using the calcium phosphate method. Overall, the highest transfection efficiencies were consistently obtained using LipofectAMINE 2000. Mol. Reprod. Dev. 63: 309–317, 2002. © 2002 Wiley‐Liss, Inc.