It has been previously reported that the carboxyl terminal residues 276 and 277 of ${alpha}$-tropomyosin are important for actin affinity. In order to investigate actin affinities of these two residues of skeletal (HA) and smooth (QT) muscle ${alpha}$-tropomyosins, a series of mutant tropomyosins were constructed in which residues at either 276 or 277 were individually replaced with a cysteine residue for chemical modification. These mutants were overexpressed in E. coli as unacetylated and Ala-Ser (AS) dipeptide fusion forms. While actin affinities of unacetylated tropomyosins were considerably low, those of AS/TMs were remarkably higher than those of corresponding unacetylated tropomyosins. However, actin affinities of AS/TM24 (QC) and AS/TM29 (HC) were dramatically lower than those of other AS/TMs and were close to those of unacetylated tropomyosins. In addition, actin affinities of unacetylated TM24 (QC) and TM29 (HC) failed to be restored in the presence of troponin, unlike unacetylated TM10 (HA) and TM23 (CA). These results indicated that the presence of a cysteine residue at 277 caused a drastic decrease in actin affinity, and also that the residue 277 is important for actin affinity of ${alpha}$-tropomyosin. Since TM23 (CA) showed high actin affinity, it may serve as a valuable tool for chemical modification studies for investigating the interaction of the carboxyl terminal residues of ${alpha}$-tropomyosin with actin and/or troponin.