Monoclonal antibodies (MAbs) specific for Pseudomonas pseudomallei antigens were produced by immunizing BALB/c mice with a crude whole cell extract. Hybrids secreting MAbs specific for P. pseudomallei antigens were identified by an indirect enzyme-linked immunosorbent assay (ELISA) against a panel of crude whole cell extracts from P. pseudomallei, P. cepacia, P. aeruginosa, P. putida, P. alcaligenes, Xanthomonas maltophilia, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Salmonella typhi, S. krefeld, S. enteritidis, Proteus mirabilis, and Staphylococcus aureus. Of the six specific clones, clone 5F8, which was IgM-producing and which reacted with all 56 P. pseudomallei isolates, was selected for further characterization and evaluation of its possible diagnostic potential. Results obtained from the indirect ELISA against various P. pseudomallei antigens, from direct bacterial agglutination, and from immunofluorescence tests suggested that 5F8 reacted with the surface envelope, and probably specifically with an epitope of the lipopolysaccharide. The antibody could be readily used to identify P. pseudomallei in primary culture or in a simulated hemoculture. The antibody was also used to prepare an affinity-purified antigen for use in an indirect ELISA that was highly sensitive and specific for the detection of circulating antibody in patients with acute septicemic melioidosis.