Improved strategies for oil-spill remediation will follow a better understanding of the nature, activities and regulating parameters of petroleum hydrocarbon-degrading microbial communities in temperate marine environments. The addition of crude oil to estuarine water resulted in an immediate change in bacterial community structure, increased abundance of hydrocarbon-degrading microorganisms and a rapid rate of oil degradation, suggesting the presence of a pre-adapted oil-degrading microbial community and sufficient supply of nutrients. Relatively rapid degradation was found at 4°C, the lowest temperature tested; and it was temperature rather than nutrient addition that most influenced the community structure. A detailed phylogenetic analysis of oil-degrading microcosms showed that known hydrocarbonoclastic organisms like Thalassolituus and Cycloclasticus, as well as proposed oil degraders like Roseobacter, were present at both 4°C and 20°C, demonstrating the thermo-versatility of such organisms. Clones related to Oleispira antarctica (98% 16S rRNA similarity), a psychrophilic alkane degrader, were dominant in the 4°C oil-degrading community, whereas other clones constituting a different clade and showing 94% similarity 16S rRNA with O. antarctica were found in situ. These findings demonstrate the potential for intrinsic bioremediation throughout the course of the year in temperate estuarine waters, and highlight the importance of both versatile psychrotolerant and specialized psychrophilic hydrocarbon-degrading microbes in effecting this process at low temperatures.