A single-point mutation in the gene encoding the ventricular myosin regulatory light chain (RLC) is sufficient to cause familial hypertrophic cardiomyopathy (FHC). Most likely, the underlying cause of this disease is an inefficient energy utilization by the mutated cardiac muscle. We set out to devise a simple method to characterize two FHC phenotypes caused by the R58Q and D166V mutations in RLC. The method is based on the ability to observe a few molecules of actin in working ex vivo heart myofibril. Actin is labeled with extremely diluted fluorescent dye, and a small volume within the I-band (∼10-16 L), containing on average three actin molecules, is observed by confocal microscopy. During muscle contraction, myosin cross-bridges deliver cyclic impulses to actin. As a result, actin molecules undergo periodic fluctuations of orientation. We measured these fluctuations by recording the parallel and perpendicular components of fluorescent light emitted by an actin-bound fluorophore. The histograms of fluctuations of fluorescent actin molecules in wild-type (WT) hearts in rigor were represented by perfect Gaussian curves. In contrast, histograms of contracting heart muscle were peaked and asymmetric, suggesting that contraction occurred in at least two steps. Furthermore, the differences between histograms of contracting FHC R58Q and D166V hearts versus corresponding contracting WT hearts were statistically significant. On the basis of our results, we suggest a simple new method of distinguishing between healthy and FHC R58Q and D166V hearts by analyzing the probability distribution of polarized fluorescence intensity fluctuations of sparsely labeled actin molecules.