Summary In the framework of a European project entitled “High Resolution Automated Microbial Identification”, the ability of rRNA gene restriction pattern determination (ribotyping) to uncover the taxonomic diversity of a collection of bacteria was tested on 226 strains of Pseudomonas sensu lato. Improved ribotyping methodology included automated DNA extraction and purification, low voltage electrophoresis, vacuum transfer of restricted, DNA fragments to nylon membranes, hybridization with a chemically labeled probe in a hybridization incubator, computer capture of restriction patterns with automatic migration measurements, fragment size interpolation, and comparison of patterns using programs of the Taxotron package (Institut Pasteur, Paris, France). Two endonucleases were used (SmaI and HincII). The strains, belonging to more than 40 nomenspecies, displayed 169 unique rRNA gene restriction patterns with SmaI, and 159 unique rRNA gene restriction patterns with HincII. A combined analysis of both restriction data yielded 79 ribogroups or isolated ribotypes. Most (92.7%) ribogroups were homogeneous with respects to nomenspecies. Some nomenspecies were split in several ribogroups (e.g. P. putida, P. fluorescens, P. marginalis, P. pseudoalcaligenes). Nomenspecies represented by single strains gave isolated ribotypes. Biovars of P. fluorescens and P. putida formed distinct ribogroups. However, P. marginalis pathovars were not separated by ribotyping. When the ribotyping data (ribogroups) were compared to published phenotypic data (phenons) obtained with Biotype-100 strips (BioMerieux, La Balme-les-Grottes, France) and Biolog GN Microplates (Biolog, Hayward, CA), an exclusive phenon-ribogroup correspondence was observed for 13 Biotype-100 phenonslribogroups and for 14 Biolog phenonslribogroups. Other Biotype-100 or Biolog phenons showed partial correspondence with ribogroups or were split into several ribogroups. Most unclustered strains in phenotypic analyses were also unclustered in the ribotyping study. DNA-DNA hybridization was used to verify some species delineation. Ribogroup R38 which contained most of P. fluorescens biovar I strains was found to constitute a DNA hybridization group distinct from P fluorescens strains in other ribogroups or biovars. The type strain of P fluorescens, which was unclustered by ribotyping, was found isolated by DNA relatedness. Ribogroup R1 which contained most of P. putida biovar A strains was found to constitute a DNA relatedness group distinct from biovar B strains. A total of 30 isolates from polluted environment were included in the study and 9 of them were identified to six known taxons. Thirteen other isolates constituted five ribogroups not represented by a reference strain and seven isolates gave isolated ribotypes. This work showed the identification otential of ribotyping and the need to extend the ribotype database. It is now clear that the ribotype carries taxonomic information in addition to typing information.