Thymosin beta(4) binds G-actin in a 1:1 ratio and prevents its aggregation to F-actin by sequestration. Substitution or modification of single amino acid residues within the N-terminal sequence 1 to 22 of thymosin beta(4) alters its interaction with G-actin. We generated thymosin beta(4) variants with amino acid substitutions within the N-terminal alpha-helix and the putative actin-binding motif. None of the E. coli-generated thymosin beta(4) variants was modified or acetylated at its N terminus. The stability of the complex of G-actin with nonacetylated thymosin beta(4) or beta(4)(A7V) is higher than the one with naturally occurring thymosin beta(4), which is always acetylated. The complex of G-actin with nonacetylated thymosin beta(4)(A7V,K18,19A) and beta(4)(K14,16,18,19A) is 15 times less stable compared to the complex with thymosin beta(4). The G-actin sequestering activities of all thymosin beta(4) variants correspond to their complex stabilities with G-actin, except for nonacetylated thymosin beta(4)(A7V), where it is attenuated. Thymosin beta(4)(Delta17-23) missing the putative actin-binding motif shows no interaction with G-actin.