S100A6 is a low molecular weight Ca2+-binding protein belonging to the S100 family. Many reports indicate that in the cell S100A6 has an influence on the organization of actin filaments, but so far no direct interaction between S100A6 and actin has been shown. In the present study we investigated binding of S100A6 to actin and the actin-tropomyosin complex. The analyses were performed on G- and F-actin and two tropomyosin isoforms-Tpm1.6 and Tpm1.8. Using purified proteins and a variety of biochemical approaches we have shown that, in a Ca2+-bound form, S100A6 directly interacts with G- and F-actin and with tropomyosin, preferentially with isoform Tpm1.8. S100A6 and tropomyosin bind to the same population of filaments and the presence of tropomyosin on the microfilament facilitates the binding of S100A6. By applying proximity ligation assay we have found that in NIH3T3 fibroblasts S100A6 forms complexes both with actin and with tropomyosin. These results indicate that S100A6, through direct interactions with actin and tropomyosin, might regulate the organization and functional properties of microfilaments.