AbstractInfection of host cells byListeria monocytogenesresults in the recruitment of cytoplasmic actin into a tail‐like appendage that projects from one end of the bacterium. Each filamentous actin tail progressively lengthenes, providing the force which drives the bacterium in a forward direction through the cytoplasm and later results inListeriacell‐to‐cell spread. Host cell actin monomers are incorporated into the filamentous actin tail at a discrete site, the bacterial‐actin tail interface. We have studied the consequences of microinjecting three different actin monomer‐binding proteins on the actin tail assembly andListeriaintracellular movement. Introduction of high concentrations of profilin (estimated injected intracellular concentration 11–22 m̈M) into infected PtK2 cells causes a marked slowing of actin tail elongation and bacterial migration. Lower intracellular concentrations of two other injected higher affinity monomer‐sequenstering proteins, Vitamin D‐binding protein (DBP; 1–2 m̈M) and DNase I (6–7 m̈M) completely block bacterial‐induced actin assembly and bacterial migration. The onset of inhibition by each protein is gradual (10–20 min) indicating that the mechanisms by which these proteins interfere withListeria‐induced actin assembly are likely to be complex. To exclude the possibility thatListeriarecruits preformed actin filaments to generate the tails and that these monomer‐binding proteins act by depolymerizing such performed actin filaments, living infected cells have been injected with fluorescently labeled phalloidin (3 m̈M). Although the stress fibers are labeled, no fluorescent phalloidin is found in the tails of the moving bacteria. These results demonstrate thatListeria‐induced actin assembly in PtK2 cells is the result of assembly of actin monomers into new filaments and thatListeria'sability to recruit polymerization competent monomeric actin is very sensitive to the introduction of exogenous actin monomer‐bindi