Organically bound sulfur makes up about 90% of the total sulfur in soils, with sulfonates often the dominant fraction. Actinobacteria affiliated to the genus Rhodococcus were able to desulfonate arylsulfonates in wheat rhizospheres from the Broadbalk long-term field wheat experiment, which includes plots treated with inorganic fertilizer with and without sulfate, with farmyard manure, and unfertilized plots. Direct isolation of desulfonating rhizobacteria yielded Rhodococcus strains which grew well with a range of sulfonates, and contained the asfAB genes, known to be involved in sulfonate desulfurization by bacteria. Expression of asfA in vitro increased >100-fold during growth of the Rhodococcus isolates with toluenesulfonate as sulfur source, compared with growth with sulfate. By contrast, the closely related Rhodococcus erythropolis and Rhodococcus opacus type strains had no desulfonating activity and did not contain asfA homologues. The overall actinobacterial community structure in wheat rhizospheres was influenced by the sulfur fertilization regime, as shown by specific denaturing gradient gel electrophoresis of PCR amplified 16S rRNA gene fragments, and asfAB clone library analysis identified nine different asfAB genotypes closely affiliated to the Rhodococcus isolates. However, asfAB-based multiplex restriction fragment length polymorphism (RFLP)/terminal-RFLP analysis of wheat rhizosphere communities revealed only slight differences between the fertilization regimes, suggesting that the desulfonating Rhodococcus community does not specifically respond to changes in sulfate supply.