The energy-dependent secretion of aerolysin by Aeromonas hydrophila requires the ExeA and ExeB proteins. An 85 kDa complex containing the two proteins was identified in wild-type cells but not in cells producing either protein alone. Radiolabelling followed by cross-linking, immunoprecipitation and then reduction of the cross-links confirmed the presence of the two proteins in the same complex. The complex could also be extracted intact from cell membranes with non-ionic detergents. A G229D substitution in the kinase-3a motif of ExeA strongly reduced the level of aerolysin secretion, as did the replacement of the invariant Lys of the kinase-1a motif (K56) with Arg. The G229D mutant contained very little of the ExeA-ExeB complex, but overexpression of the mutant complex until wild-type levels were achieved allowed normal secretion. In contrast, the K56R mutation had no effect on complex formation, but normal secretion levels occurred only when there was a far greater amount of the complex present. These results are consistent with a model in which binding of ATP by ExeA is required for ExeA-ExeB complex formation, while hydrolysis is required for its function in secretion once established.