This research was conducted to identify culturable surfactant-producing bacterial species that inhabit the 40,000-year-old natural asphalt seep at the Rancho La Brea Tar Pits in Los Angeles, CA. Using phenanthrene, monocyclic aromatic hydrocarbons, and tryptic soy broth as growth substrates, culturable bacteria from the tar pits yielded ten isolates, of which three species of gamma-proteobacteria produced biosurfactants that accumulated in spent culture medium. Partially purified biosurfactants produced by these strains lowered the surface tension of water from 70 to 35-55 mN/m and two of the biosurfactants produced 'dark halos' with the atomized oil assay, a phenomenon previously observed only with synthetic surfactants. Key findings include the isolation of culturable biosurfactant-producing bacteria that comprise a relatively small fraction of the petroleum-degrading community in the asphalt.