Monoclonal antibodies against the alkaline phosphatase of Pseudomonas aeruginosa were produced from spleen cells of BALB/c mice primed with purified alkaline phosphatase of P. aeruginosa ATCC 10145 and SP20/Ag-14 myeloma cells. The eight stable clones established produced antibodies that reacted by enzyme-linked immunosorbent and indirect immunofluorescence assays with all bacterial strains of P. aeruginosa, including the 17 serotypes and two nontypable strains. Three of the clones cross-reacted only with some Pseudomonas species of the rRNA homology group I defined by N. J. Palleroni (in N. R. Krieg and J. G. Holt, ed., Bergey's Manual of Systematic Bacteriology, 8th ed., p. 140-218, 1984). The other clones also interacted with other species, including Pseudomonas acidovorans and Xanthomonas maltophilia. Because other species of the genera Aeromonas and Acinetobacter and species of the family Enterobacteriaceae were not detected by these monoclonal antibodies, the antibodies could be used as reagents for routine detection of P. aeruginosa in clinical specimens. Interactions of the antibodies with other Pseudomonas species such as P. fluorescens and P. stutzeri are not important, since these species are susceptible to the same antipseudomonal agents.