The interaction of filamentous actin with alpha-actinin, an actin cross-linking protein, is well established. On the other hand, monomeric actin-alpha-actinin interaction has been a subject of controversy. In this report, we have characterized the interaction of monomeric actin, coated on plastic plates under conditions of non-polymerization, with alpha-actinin in presence of magnesium. Using specific polyclonal anti-actin antibodies, with the whole molecule or purified peptides, we have localized two sites of interaction on action molecule: one near Thr-103 and a new one in the twenty last amino acids.