Biosurfactants produced by subsurface bacteria may potentially enhance in situ bioremediation. A study was performed to characterize the production and enhancement of production of biosurfactants by 37 gasoline-degrading bacteria isolated from a gasoline-contaminated aquifer. Surface tension and emulsification capacity of the bacterial cultures growing on unleaded gasoline vapors were determined and mean surface tension and emulsification capacity values calculated. When liquid gasoline was added to the cultures, the mean surface tension reduction of the sample population was nearly doubled, compared to the cultures using only gasoline vapors. Many of the bacterial isolates were able to grow without significant surface tension reductions or emulsification. For two select isolates, optimum ammonium sulfate concentrations for growth and biosurfactant production were determined. Addition of yeast extract enhanced biosurfactant production by the two isolates. Both isolates were shown to initiate production of biosurfactants during the stationary phase of cell growth.