We describe a spontaneous rough mutant of Listeria monocytogenes that produces reduced amounts of a 60-kilodalton major extracellular polypeptide (p60) as shown by sodium dodecyl sulfate--polyacrylamide gel electrophoresis and Western blot analysis. The cells of this mutant are filamentous, do not give rise to smooth wild-type colonies, and produce listeriolysin O in amounts equal to that of the wild-type cells, but they show a reduced virulence in the mouse LD50 model and in the Caco-2 tissue culture virulence assay. Light and electron microscopic studies show that this mutant invades and remains filamentous during in vivo growth in both Caco-2 and 3T6 tissue culture monolayers. The reduced virulence of the rough mutant is not due to the inability of its filamentous forms to invade or to grow in nonprofessional phagocytes since invasion and growth of the smooth wild-type and the rough mutants are comparable in both Caco-2 and 3T6 monolayers.