Intracellular actin-based motility of Listeria monocytogenes requires protein-protein interactions involving two different proline-rich sequences: first, the tightly bound bacterial surface protein ActA uses its multiple oligoproline registers [consensus sequence = FE(D)FPPPPTD(E)E(D)] to tether vasodilator-stimulated phosphoprotein (VASP) to the bacterial surface; and second, VASP then deploys its own multiple GPPPPP (or GP5) registers to localize the actin-regulatory protein profilin to promote actin polymerization. We now report that fluorescence titration showed that GP5GP5GP5 peptide binds to profilin (KD of 84 microM), and the peptide weakly inhibits exchange of actin-bound nucleotide in the absence or presence of profilin. Microinjection of synthetic GPPPPP triplet into Listeria-infected PtK2 cells promptly arrested motility at an intracellular concentration of 10 microM. This inhibition was completely neutralized when equimolar concentrations of profilin and GP5GP5GP5 were simultaneously microinjected. Fluorescence studies with [His-133-Ser]-profilin, a site-directed mutant previously shown to be defective in binding poly-l-proline [Bjorkegren, C., Rozycki, M., Schutt, C. E., Lindberg, U., & Karlsson, R. (1993) FEBS Lett. 333, 123-126], exhibits little or no evidence of saturable GP5GP5GP5 binding. When an equimolar concentration of this [His-133-Ser]-profilin mutant was co-injected with GP5GP5GP5, the peptide's inhibitory action remained completely unaffected, indicating that GP5GP5GP5 binding to wild-type profilin represents a key step in actin-based pathogen motility. We also present a model that shows how the focal binding of VASP with its GPPPPP registers can greatly increase the local concentration of profilin and/or profilin-actin-ATP complex at the bacteria/rocket-tail interface.