The genus Pseudomonas is one of the best-studied bacterial groups in soil, and includes numerous species of environmental interest. Pseudomonas species play key roles in soil, for instance in biological control of soil-borne plant pathogens and in bioremediation of pollutants. A polymerase chain reaction-denaturing gradient gel electrophoresis system that specifically describes the diversity of Pseudomonas spp. in soil was developed. On the basis of this molecular method as well as cultivation-based approaches, the diversity of Pseudomonas species in soil under different agricultural regimes (permanent grassland, arable land either under rotation or under monoculture of maize) was studied. Both types of approaches revealed differences in the composition of Pseudomonas populations between the treatments. Differences between the treatments were also found based on the frequency of isolation of Pseudomonas strains with antagonistic properties against the soil-borne pathogen Rhizoctonia solani AG3. Higher relative numbers of isolates either with antagonistic activity toward this pathogen or with chitinolytic activity were obtained from permanent grassland or from the short-term arable land than from the arable land. The results obtained in this study strongly indicate that agricultural regimes influence the structure of Pseudomonas populations in soil, with specific antagonistic subpopulations being stimulated in grassland as compared to arable land.