Actin filament subunit interfaces are required for the proper interaction between filamentous actin (F-actin) and actin binding proteins (ABPs). The production of small F-actin complexes mimicking such interfaces would be a significant advance toward understanding the atomic interactions between F-actin and its many binding partners. We produced actin lateral dimers and trimers derived from F-actin and rendered polymerization-deficient by ADP-ribosylation of Arg-177. The degree of modification resulted in a moderate reduction in thermal stability. Calculated hydrodynamic radii were comparable to theoretical values derived from recent models of F-actin. Filament capping capabilities were retained and yielded pointed-end dissociation constants similar those of wild-type actin, suggesting native or near-native interfaces on the oligomers. Changes in DNase I binding affinity under low and high ionic strength suggested a high degree of conformational flexibility in the dimer and trimer. Polymer nucleation activity was lost upon ADP-ribosylation and rescued upon enzyme-mediated deADP-ribosylation, or upon binding to gelsolin, suggesting that interactions with actin binding proteins can overcome the inhibiting activities of ADP-ribosylation. The combined strategy of chemical cross-linking and ADP-ribosylation provides a minimalistic and reversible approach to engineering polymerization-deficient F-actin oligomers that are able to act as F-actin binding protein scaffolds.