Petroleum degradation studies are being done to obtain a seasonal incidence, as well as species distribution of petroleum-degrading microorganisms in Chesapeake Bay. From analysis of water and sediments collected at two stations in Chesapeake Bay it was found that the concentration of petroleum in an oil polluted site in Baltimore Harbor was five times greater than in Eastern Bay. The numbers of petroleum-degrading microorganisms, measured by direct and replica plating, in the water and sediment samples were related to the concentration of oil in each sample. Total yields of petroleum-degrading microorganisms grown on an oil substrate were greater for those organisms exposed to oil in the natural environment. Microorganisms isolated from water and sediment samples collected in Baltimore Harbor grew on substrates representative of the aliphatic, aromatic and refractory hydrocarbons. From analyses of species distribution, it was observed that a hydrocarbon-utilizing fungus, Cladosporium resinae, and actinomycetes were predominant among the hydrocarbon-utilizing isolates.