Mutants of Listeria monocytogenes were recently isolated which are impaired in the synthesis of a major extracellular protein (p60). As shown in this investigation, the p60 mutants have lost the capability of invading nonprofessional phagocytic 3T6 mouse fibroblast cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of supernatant proteins of these mutants indicated that no other extracellular protein was altered in these mutants. The p60 mutants formed long cell chains which disaggregated to normal-sized single bacteria upon treatment with partially purified p60. These disaggregated bacterial cells were able to invade 3T6 cells. Physical disruption of the cell chains by ultrasonication produced similar single cells which were, however, noninvasive. Treatment of these ultrasonicated mutant cells with wild-type p60 restored their ability to invade 3T6 cells.