Biosurfactants have potential for enhancement of in situ biorestoration. A study was performed to characterize biosurfactant production by microbes from several subsurface environments contaminated with petroleum hydrocarbons. Emulsification of hydrocarbons was used to identify microorganisms with the greatest potential to produce biosurfactants. Biostimulated zone microbes contained the greatest proportion of emulsifiers as compared to contaminated and pristine zone microbes. Biostimulation was the dominant factor which selected for and encouraged growth of microbial emulsifiers; exposure to hydrocarbon was also important. The degree of emulsification by biostimulated microbes was greater for the contaminant hydrocarbon than for heavier fuels. Emulsification of hydrocarbons may be an important mechanism in biorestoration by increasing the bioavailability of the contaminant; however, no attempt was made to assess this possibility or relationship. Since biosurfactants are emulsion stabilizers, they merit further attention for enhancement of in situ aquifer restoration.