SummaryIntercellular spreading of shigellae Is a prerequisite for shigellosis, although the molecular mechanisms underlying the phenomenon are still largely obscure. To elucidate some of these mechanisms, we performed random TniO insertion mutagenesis inShigella flexneriYSH6000T and found a chromosomal locus in the Notl‐J segment responsible for bacterial spreading. The locus affected in the mutant, designatedvacJ, was neither involved in the invasion of epithelial cells nor in intracellular movement, but was required for intercellular spread. ThevacJmutant was capable of forming bacterium‐containing membranous protrusions within the infected cell, but had diminished ability to move from the protrusions into the cytoplasm of the adjacent epithelial cells. Cloning and sequencing of thevacJregion Indicated that thevacJgene encoded a 28.0 kDa protein possessing a signal peptide at the N‐terminus, which contained the motif characteristic of lipoproteins. The analysis of thevacJproduct indicated that VacJ was exposed on the bacterial surface. ThevacJgene was distributed among shigellae and enteroinvasiveEscherichia coli, and the constructedvacJmutants failed to spread intercellularly, indicating thatvacJis a chromosomal gene essential for the pathogenicity of shig