Genetic and molecular data now available on the pathogenic properties of Shigella flexneri allow rational design of live attenuated vaccine strains. The genes required at given steps of the infection process can be selectively mutated to impair the bacterium's capacity to interact with intestinal epithelial cells and/or survive within intestinal tissues in general. We have tested two mutations in S. flexneri serotype 5a (M90T) which, alone or in combination, have yielded promising results when evaluated as vaccine prototypes in orally infected macaque monkeys. The first mutation, icsA, blocks intracellular and cell-to-cell spread of the micro-organism. This mutant (SC560) appeared reasonably well tolerated and elicited protection against homologous challenge. The second mutation, ompB, disconnects the bacterium from one of its major environmental regulatory factors, osmolarity. This mutant (SC433) still caused slight dysenteric symptoms in vaccinees. It was also perfectly protective. When these two mutations were combined, the double mutant (SC445), was perfectly tolerated but failed to protect one out of five animals. These studies bring interesting prospects of the possibility of immunizing against shigellosis. In addition to providing new possibilities for vaccine design, construction and evaluation of these mutants allowed substantial progress in understanding the pathogenesis of shigellosis.