Amid rising fuel costs, emergent environmental issues, and mounting international security concerns, considerable attention has recently returned to reviving biologically derived n-butanol as a renewable transportation fuel alternative. n-Butanol boasts physical and thermodynamic properties which are akin to gasoline and are in many ways superior to those of biologically derived ethanol. The natural n-butanol fermentation pathway in Clostridium competes with several other native pathways for metabolite fluxes which can thereby reduce its final yield. Furthermore, the cytotoxic effects of n-butanol contribute to low titers in natural organisms due to feedback inhibition. Accordingly, there is much interest in designing robust, alternative microorganisms capable of producing n-butanol. Through metabolic engineering efforts we have explored the potential for n-butanol production in a variety of bacterial hosts, including E. coli, P. putida, and B. subtilis. Within these hosts, we have explored both the reconstruction of the natural Clostridium pathway as well as the use of homologous genes from a variety of source organisms. The development of alternative hosts for the heterologous n-butanol pathway reveals important implications for both production and product tolerance.