To isolate and characterize novel thermophilic bacteria capable of biodesulfurization of petroleum. A culture containing two Paenibacillus spp. (denoted "32O-W" and "32O-Y") was isolated by repeated passage of a soil sample at up to 55 A degrees C in medium containing dibenzothiophene (DBT) as sulfur source. Only 32O-Y metabolized DBT, apparently via the 4S pathway; maximum activity occurred from 40 to 45 A degrees C, with some activity up to at least 50 A degrees C. 32O-W enhanced DBT metabolism by 32O-Y (by 22-74 % at 40-50 A degrees C). With sulfate as sulfur source, 32O-Y and 32O-W grew well up to 58 and 63 A degrees C, respectively. Selection of a mixed culture of 32O-Y and 32O-W at 54 A degrees C increased DBT metabolism 36-42 % from 40 to 45 A degrees C. Genome sequencing identified desulfurization gene homologs in the strains consistent with their desulfurization properties. The 32O-Y/32O-W culture may be a useful starting point for development of an improved thermophilic petroleum biodesulfurization process.