A heterologous, noncompetitive, sandwich, enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of Aeromonas salmonicida. This paper reports the initial, laboratory validation of the applications of this assay to the study of the ecology of A. salmonicida in the effluents of freshwater hatcheries, marine sediments and in fish. The selectivity of the assay was investigated by the use of four (one core and three application-dependent) control panels of bacteria. No false-negative and two false-positive reactions were detected in the core panel that contained 108 strains of the target species, 100 strains of nontarget mesophilic aeromonads and 19 miscellaneous strains. The two false-positive reactions were generated by strains of Staphylococcus aureus. In the 74-strain panel for application to fish tissues and the 150 strain panel for freshwater sediment applications, no false positives were detected. Two false positives were, however, detected in the 150-strain panel for applications to marine sediments. Nine samples of environmental matrices, which the proposed applications of this assay might require to be examined, were used in an investigation of performance characteristics of the assay in spiked and unspiked matrices. The matrices exerted a slight influence of the baseline values generated by the assay and reduced the intra- and interassay precision; however, in all cases, these changes were within acceptable limits. The applied lower detection limits for A. salmonicida in spiked environmental matrices were in the range 1-4 x 10(3) cfu/ml and were slightly higher than the limits detected in broth suspensions of the organism. The ELISA was also demonstrated to have the ability to detect A. salmonicida in studies of incurred freshwater and sediments. The laboratory experiments reported here failed to detect any evidence that would argue against the initiation of full-scale validation studies of this method under field conditions. (C) 2003 Elsevier Science B.V. All rights reserved.