The interaction of recombinant human small heat shock protein with apparent molecular mass 20 kDa (Hsp20, HspB6) with actin was investigated. Wild type Hsp20 and its S16D mutant mimicking phosphorylation of Hsp20 by cyclic nucleotide-dependent protein kinases do not affect the rate and extent of actin polymerization. Ultracentrifugation of the mixture of Hsp20 (or its S16D mutant) with isolated F-actin or F-actin containing tropomyosin, calponin or alpha-actinin resulted in co-sedimentation of less than 0.04 mol of Hsp20 monomer per mol of actin. Myofibrils of skeletal, cardiac or smooth muscle bound less than 0.04 mol of Hsp20 monomer per mol of actin and this stoichiometry was independent of phosphorylation or mutation of Ser16 of Hsp20. Since Hsp20 is not a genuine actin-binding protein, the earlier described correlation between Hsp20 phosphorylation and smooth muscle relaxation cannot be explained by direct interaction of Hsp20 with actin.