Actin is the major cytoskeletal protein of virtually all eukaryotic cells. Actin assembly/disassembly is involved in a variety of cellular processes and actin-binding proteins are essential in regulation of the pool of actin monomers. Cofilin and DNase I are actin-binding proteins, which form both binary (actin-DNase 1, cofilin-actin) and ternary (cofilin-actin-DNase I) complexes with actin. Here we use native gel electrophoresis to examine the roles of ATP, ADP, Ca2+ and Mg2+ in the formation of these complexes as well as on the ability of actin to self-assemble. Conditions which favour actin polymerisation are: ATP (no Me2+) greater than or equal to ADP (no Me2+) > ADP-Ca2+ = ADP-Mg2+ > ATP-Mg2+ 1 ATP-Ca2+. Preferential conditions for the formation of the binary actin-cofilin complex are: ADP-Mg2+ greater than or equal to ADP-Ca2+ >> ATP-Ca2+ approximate to ATP-Mg2+ approximate to ADP-No Me2+ approximate to ATP-No Me2+. Actin forms a very tight complex with DNase I in the order: ATP-Ca2+ greater than or equal to ATP-Mg2+ approximate to ADP-Mg2+ approximate to ADP-Ca2+ greater than or equal to ADP-(no Me2+) > ATP-(no Me2+). Effectively, the complex does not form in the presence of ATP and the absence of free Me2+. Finally, the conditions which favour the formation of a ternary complex of cofilin-actin-DNase I resemble the actin-DNase I, namely: ATP-Ca2+ approximate to ADP-Ca2+ approximate to ADP-Mg2+ approximate to ATP-Mg2+ approximate to ADP (no Me2+)> ATP-(no Me2+).