The Listeria monocytogenes surface protein ActA is an important virulence factor that plays an essential role in intracellular movement of Listeria cells by inducing actin polymerisation. The ActA protein is known to interact with several mammalian proteins including the phosphoprotein VASP, actin and the Arp2/3 complex. In a search for additional ActA-binding proteins we recently employed the yeast two-hybrid system to search for proteins that interact with ActA, and identified, among others, the mammalian protein LaXp180 as a binding partner. In the present study the interaction of the two proteins was investigated in more detail. A number of variants were tested in the yeast two-hybrid system for their ability to interact. On the basis of these assays, the 14 C-terminal amino acids of LaXp180 were identified as being necessary for the interaction with ActA. The proline-rich repeat (PRR) region of ActA was found to be necessary for the interaction with LaXp180, but upstream or downstream sequences are also required to enhance the specificity of the interaction. The second and third repeats in ActA are especially important, and the minimal sequence of ActA capable of interacting with LaXp180 was a proline- and glutamate-rich stretch of PRR3 fused to part of the N-terminal sequence of ActA. Further analysis using site-specific mutations located in either the C-terminal region of LaXp180 or the proline-rich motif of PRR3 of ActA showed that three positively charged amino acids in LaXp180 and two negatively charged amino acids in ActA are critical for the interaction of the two proteins.