Cytotoxic enterotoxin (Act) is a key virulence factor in the pathogenesis of infections caused by Aeromonas spp. The cytotoxic enterotoxin gene (act) was detected in 32 out of 69 environmental isolates of Aeromonas spp. by hybridization with the act gene probe. To evaluate the pathogenic potential of the act gene probe-positive isolates, 32 act gene probe-positive and 31 randomly selected act gene probe-negative isolates were tested for enterotoxicity in a suckling mice assay (SMA), for haemolytic activity on sheep blood agar plates, for the presence of CAMP-like factors, and for cytotoxicity in a Vero cell line. The act gene probe-positive isolates significantly differed from the toxin gene probe-negative ones with respect to enterotoxicity in the SMA (P=0·009) and haemolytic activity (P=0·005). The CAMP–haemolysin phenotype was significantly associated with the rabbit ileal loop assay (P=0·08), Vero cell assay (P=0·064), and haemolysin production under the microaerophilic conditions (P=0·056) of the act gene probe-positive isolates of Aeromonas spp. These data indicated the role of Act in the pathogenesis of Aeromonas infections and that the enterotoxic potential of Aeromonas spp. could be assessed by simply performing a CAMP–haemolysin assay.