SUMMARY: Sheared polar flagella of the type strains of monotrichate Pseudomonas aeruginosa and multitrichate P. fluorescens and P. putida were purified and the sedimentation coefficients of the corresponding flagellins determined by ultracentrifugation; from these values the approximate molecular weights of the major flagellins were calculated to be between 38000 and 43000, and other estimates based on methionine content also suggested that the range was between 38800 and 43000. Purified flagellins of these three species, and also P. syringae pv. morsprunorum and Pseudomonas sp. (‘Vibrio percolans’), were digested with trypsin, and the peptide maps obtained after two-dimensional electrophoresis were compared. Altogether 65 peptide spots were delineated and 15 peptides were found to be common to all five species. The amino acid composition of the purified flagellins of the three type species was found to accord qualitatively and quantitatively with other prokaryote flagellins. Antisera were raised against the three purified flagellins, and tested by the Ouchterlony diffusion plate method against (a) purified homologous and heterologous flagellins, (b) deflagellated cells with sheared flagella and (c) crude sheared flagella of a further 67 test strains and species (mainly Pseudomonas). With either flagella or flagellin antigens the serological reactions were virtually identical, but not all strains of a given species reacted with the relevant antiflagellin antiserum. Rapid species identification using a single antiflagellin antiserum was thus not possible. The flagellar location of the antigenic site(s) was confirmed by two serological methods, including direct immunofluorescent serology.