Histidine residues of actin were carbethoxylated with diethylpyrocarbonate. Four and three histidine residues per actin monomer were rapidly carbethoxylated in the G and F form, respectively. After partial Carbethoxylation of G-actin a non-polymerizable fraction could be separated by ultracentrifugation. The analysis of these fractions showed that one of the four fast-reacting histidines of G-actin plays an essential role in polymerization. Removal of the carbethoxy groups from the histidine residues by hydroxylamine treatment restored the ability to polymerize. The specificity of the reaction was tested by the use of [14C]diethylpyrocarbonate. After tryptic digestion of non-polymerizable carbethoxylated actin a single histidyl peptide was isolated by gel filtration and diagonal electrophoresis. The histidine residue essential for polymerization was identified as histidine-40.