The Renewable Fuel Standard (RFS2) under the Energy Independence and Security Act of 2007 requires 15.2 billion gallons of domestic alternative fuels per year by 2012, of which 2 billion gallons must be from advanced biofuel and emit 50% less life-cycle greenhouse gas (GHG) emissions than petroleum-based transportation fuels. Microalgal biodiesel, one type of advanced biofuel, has the qualities and potential to meet the RFS's requirement. A comparative life cycle assessment (LCA) of four microalgal biodiesel production conditions was investigated using a process LCA model with Monte Carlo simulation to assess global warming potential (GWP), eutrophication, ozone depletion and ecotoxicity potentials. The four conditions represent minimum and maximum production efficiencies and different sources of carbon dioxide and nutrient resources, i.e. synthetic and waste resources. The GWP results of the four CO2 microalgal biodiesel production conditions showed that none of the assumed production conditions meet the RFS's GHG requirement. The GWP results are sensitive to energy consumption in harvesting process. Other impacts such as eutrophication, ozone depletion and ecotoxicity potentials, are sensitive to percent lipid content of microalgae, service lifetime of PBRs and quantity of hexane in extraction process, respectively. Net energy ratio and other emissions should be included in future RFS for a more sustainable fuel.