The functional properties of wild type α-tropomyosin expressed in E. coli with an alanine-serine N-terminal leader (AS-α-Tm) were compared with those of AS-α-Tm with either of two missense mutations (Asp175Asn and Glu180Gly) shown to cause familial hypertrophic cardiomyopathy (FHC). Wild type AS-α-Tm and AS-α-Tm(Asp175Asn) binding to actin was indistinguishable from rabbit skeletal muscle ab-tropomyosin whilst the affinity of AS-α-Tm (Glu180Gly) was about threefold weaker.In vitromotility assays were performed with AS-α-tropomyosin incorporated into skeletal muscle actin-rhodamine phalloidin filaments moving over skeletal muscle heavy meromyosin. Under relaxing conditions (pCa9), troponin added to actin filaments containing AS-α-tropomyosin or mutant tropomyosins resulted in normal switch-off, with a decrease in the fraction filaments moving from >80% to <20%. Under activating conditions (pCa5), troponin had a minor effect upon actin-AS-α- tropomyosin filament velocity (increased by 5+/-1%, n=10), whereas the velocity increased by 18+/-3%(n=7) with actin filaments containing AS-α-tropomyosin(Asp175Asn) and by 21+/-2%(n=8) with filaments containing AS-α-tropomyosin(Glu180Gly) (p<0.05 compared with AS-α- tropomyosin). Thus FHC mutations in α-tropomyosin produce detectable changes in the Ca 2 + - regulation of thin filaments, presumably via altered interaction with troponin.