To identify the microorganisms involved in benzene degradation, DNA-stable isotope probing (SIP) with ¹³C-benzene was applied to a methanogenic benzene-degrading enrichment culture. Pyrosequencing of ribosomal RNA (rRNA) gene sequences revealed that the community structure was highly complex in spite of a 3-year incubation only with benzene. The culture degraded 98% of approximately 1 mM ¹³C-benzene and mineralized 72% of that within 63 d. The terminal restriction fragment length polymorphism (T-RFLP) profiles of the buoyant density fractions revealed the incorporation of ¹³C into two phylotypes after 64 d. These two phylotypes were determined to be Desulfobacterales- and Coriobacteriaceae-related bacteria by cloning and sequencing of the 16S rRNA gene in the ¹³C-labeled DNA abundant fraction. Comparative pyrosequencing analysis of the buoyant density fractions of ¹²C- and ¹³C-labeled samples indicated the incorporation of ¹³C into three bacterial and one archaeal OTUs related to Desulfobacterales, Coriobacteriales, Rhodocyclaceae, and Methanosarcinales. The first two OTUs included the bacteria detected by T-RFLP-cloning-sequencing analysis. Furthermore, time-resolved SIP analysis confirmed that the activity of all these microbes appeared at the earliest stage of degradation. In this methanogenic culture, Desulfobacterales- and Coriobacteriaceae-related bacteria were most likely to be the major benzene degraders.