Of eight laboratory cultures of marine gamma- and alpha-Proteobacteria tested, growth on glycolate as a sole carbon source was detected for only three species: Pseudomonas stutzeri, Oceanimonas doudoroffii and Roseobacter sp. isolate Y3F. Degenerate polymerase chain reaction (PCR) primers were designed to amplify glcD, which encodes the D-subunit of the enzyme glycolate oxidase; glcD could be amplified only from those cultures that grew on glycolate. The PCR primers were used to explore glcD diversity in four field samples collected from different ocean environments: an Atlantic Gulf Stream Ring, sampled above and below the thermocline and two Pacific coastal sites, Parks Bay and San Juan Channel, WA. Environmental glcD sequences belonged to six major bacterial phylogenetic groups, with most sequences forming novel clades with no close relatives. Different patterns of glcD diversity were observed within and between the two nutrient regimes. Comparison of glcD and 16S rDNA diversity and analyses of available bacterial genomes and a metgenomic library from the Sargasso Sea show that glycolate-utilizing potential exists in only a subset of bacteria. Glycolate is produced in marine environments mainly by phytoplankton. Examination of glcD diversity will aid in understanding the influence of phytoplankton on bacterial community structure.