Gelsolin binds two monomers in the nucleating complex with G-actin in calcium and caps actin filaments. However, 3 actin-binding domains have been identified within its 6 repeating sequence segments corresponding to S1 S2-3 and S4-6. S1 and S4-6 bind only G-actin whereas S2-3 binds specifically to F-actin. Two of the three domains (S2-3 and S4-6) are required for nucleation and a different pair (S1 and S2-3) for severing. Here we show for the first time that the domains unique to nucleation (S4-6) or severing (S1) compete for the same region on subdomain 1 of G-actin. We further show that S2-3 binds actin monomers weakly in G-buffer conditions and that this interaction persists when S1 or S4-6 are also bound. Thus gelsolin associates with two distinct regions on actin. Since S2-3 does not bind monomeric actin in F-buffer, we suggest that its high affinity 1:1 stoichiometry for filament subunits reflects interaction with two adjacent subunits.