The thermostable direct haemolysin (TDH) is considered to be the major virulence factors of Vibrio parahaemolyticus; however, poor information is available about its mechanism of action. In our study we examined the capacity of two V. parahaemolyticus TDH-producers (strains 2067 and 3305) to induce fluid secretion in rat ileal loop and to reveal the role of calcium ions (Ca(2+)), calmodulin (CaM), and protein kinase C (PKC) in V. parahaemolyticus TDH(+)-induced fluid secretion. The results show that V. parahaemolyticus TDH(+) strains were able to induce secretion in small intestine; on the contrary, this ability was not evidenced in the V. parahaemolyticus TDH(-) strain used as negative control. The data suggest an enterotoxic activity of haemolysin. Calcium ionophore A23187 and 1-verapamil (calcium channel blocker), when injected alone, induced fluid accumulation in the control loops. A further increase in fluid accumulation (P<0.001) was noted when calcium ionophore was injected along with bacterial suspension of both TDH(+) strains and a significant decrease (P<0.001) in experimental loops when 1-verapamil was inoculated along with bacterial suspension. The other modulating agents increased fluid accumulation in both control and experimental loops, without significant differences with respect to the positive control. Our findings suggest that Ca(2+) appears to be an important messenger involved in the stimulation of intestinal secretion, contrary to PKC and calmodulin which do not appear to have any role.