Several mutations in the beta-myosin heavy chain gene cause hypertrophic cardiomyopathy. This study investigates (1) the in vitro velocities of translocation of fluorescently-labelled actin by beta-myosin purified from soleus muscle of 30 hypertrophic cardiomyopathy patients with seven distinct beta-myosin heavy chain gene mutations: Thr124Ile, Tyr162Cys, Gly256Glu, Arg403Gln, Val606Met, Arg870His, and Leu908Val mutations; and (2) motility activity of beta-myosin purified from cardiac and soleus muscle biopsies in the same patients. The velocity of translocation of actin by beta-myosin purified from soleus or cardiac muscle of 22 normal controls was 0.48 +/- 0.09 micron s-1. By comparison, the motility activity was reduced in all 30 patients with beta-myosin heavy chain gene mutations (range, 0.112 +/- 0.041 to 0.292 +/- 0.066 micron s-1. Notably, the Tyr162Cys and Arg403Gln mutations demonstrated significantly lower actin sliding velocities: 0.123 +/- 0.044, and 0.112 +/- 0.041 micron s-1, respectively. beta-myosin purified from soleus muscle from four patients with the Arg403Gln mutation had a similar actomyosin motility activity compared to beta-myosin purified from their cardiac biopsies (0.127 +/- 0.045 micron s-1 versus 0.119 +/- 0.068 micron s-1, respectively). Since these seven mutations lie in several distinct functional domains, it is likely that the mechanisms of their inhibitions of motility are different.