Aims: Potential virulence factors produced by culture filtrates of Plesiomonas shigelloides isolated from water were investigated. Methods and Results: Culture filtrates of P. shigelloides strains were assayed for cytotoxic activity in CHO (Chinese hamster ovary), Vero (African green monkey kidney), HeLa (human cervix), HT29 (human epithelial intestinal) and SK6 (swine epithelial kidney) cells. Microscopic analyses revealed intensive cytoplasmic vacuolation including cell rounding and swelling, with gradual destruction of the monolayer in filtrate-treated cells. Neutral red assays showed that CHO, HeLa and Vero cells were the most sensitive to the vacuolating activity, which was evident within 30 min of culture filtrate exposure. This activity was inactived by heating at 56degreesC for 15 min and partially neutralized by antiserum to the cytotoxin of Aeromonas hydrophila . All P. shigelloides strains had a cell-associated haemolysin in the agar plate assay. Three isolates were found to produce a cell-free haemolytic activity at 37degreesC. In the suckling mouse test, two P. shigelloides culture supernatants were positive for enterotoxic activity. Conclusions: P. shigelloides culture filtrates isolated from aquatic environment cause intracellular vacuolation on mammalian cells, and produce haemolytic and enterotoxic activities. Significance and Impact of the Study: This work revealed the presence of putative virulence factors that could be associated with human infections involving Plesiomonas strains.