Proteorhodopsin (PR), a photoactive proton pump containing retinal, is present in approximately half of all bacteria in the ocean, but its physiological role is still unclear, since very few strains carrying the PR gene have been cultured. The aim of this work was to characterize PR diversity in a North Sea water sample, cultivate a strain representative of North Sea PR clusters, and study the effects of light and carbon concentration on the expression of the PR gene. A total of 117 PR sequences, of which 101 were unique, were obtained from a clone library of PCR-amplified PR gene fragments. Of the North Sea PRs, 97% were green light absorbing, as inferred from the amino acid at position 105; 67% of the PR protein fragments showed closest similarity to PRs from Alphaproteobacteria, 4% showed closest similarity to PRs from Gammaproteobacteria, and 29% showed closest similarity to PRs from “Bacteroidetes”/Flavobacteria. The dominant PR cluster (comprising 18% of all PRs) showed a high degree of similarity to the PR from the cultivated Roseobacter strain HTCC2255. The relative abundances of the North Sea PR clusters were confirmed by quantitative PCR. They were detected in metagenomic fragments from coastal oceans worldwide with various degrees of abundance. Several hundred bacterial strains from the North Sea water sample were cultivated on oligocarbophilic media. By screening with degenerate primers, two strains carrying the PR gene were identified. Their 16S rRNA gene sequences were identical and affiliated with a Bacteroidetes subcluster from the North Sea. The PR sequence of isolate PRO95 was completed by chromosomal walking. It was 76% identical to that of Dokdonia donghaensis MED134 and was functional, as indicated by the signature amino acids. PRO95 expressed its PR gene in liquid media containing between 9.7 and 121 mM carbon, both in the light and in the dark. Growth was not enhanced by light. Thus, the detection of the physiological role of PR may require more sensitive methods.