A key advantage of using microalgae for biofuel production is the ability of some algal strains to thrive in waters unsuitable for conventional crop irrigation such as saline groundwater or seawater. Nonetheless, the availability of sustainable water supplies will provide significant challenges for scale-up and development of algal biofuels. We conduct a partial techno-economic assessment based on the availability of freshwater, saline groundwater, and seawater for use in open pond algae cultivation systems. We explore water issues through GIS-based models of algae biofuel production, freshwater supply (constrained to less than 5% of mean annual flow per watershed) and costs, and cost-distance models for supplying seawater and saline groundwater. We estimate that, combined, these resources can support 9.46 × 10(7) m(3) yr(-1) (25 billion gallons yr(-1)) of renewable biodiesel production in the coterminous United States. Achievement of larger targets requires the utilization of less water efficient sites and relatively expensive saline waters. Despite the addition of freshwater supply constraints and saline water resources, the geographic conclusions are similar to our previous results. Freshwater availability and saline water delivery costs are most favorable for the coast of the Gulf of Mexico and Florida peninsula, where evaporation relative to precipitation is moderate. As a whole, the barren and scrub lands of the southwestern U.S. have limited freshwater supplies, and large net evaporation rates greatly increase the cost of saline alternatives due to the added makeup water required to maintain pond salinity. However, this and similar analyses are particularly sensitive to knowledge gaps in algae growth/lipid production performance and the proportion of freshwater resources available, key topics for future investigation.