Left Ventricular (LV) myocytes were isolated from 15-wk-old male mice bearing the Arg⁴⁰³ → Gln α-cardiac myosin heavy chain missense mutation (α-MHC^403/+), a model of familial hypertrophic cardiomyopathy. LV myocytes were classified morphologically: type I, rod shaped with parallel myofibrils; type II, irregularly shaped, shorter and wider than wild-type (WT) control cells, with parallel myofibrils; and type III, irregularly shaped with disoriented myofibrils. Compared with WT myocytes, α-MHC^403/+ myocytes had fewer type I cells (WT = 74 ± 3%, α-MHC^403/+ = 41 ± 4%,P < 0.01) and more type III cells (WT= 12 ± 3%, α-MHC^403/+ = 49 ± 7%, P < 0.01). In situ histology also demonstrated marked myofibrillar disarray in the α-MHC^403/+ hearts. With the use of video edge detection, myocytes were paced at 1 Hz (37°C) to determine the effects of the mutation on myocyte function. End-diastolic length was reduced in mutant myocytes, but fractional shortening (% contraction) and sarcomere length were not. Velocity of contraction (−dL/dt _max) was depressed in mutant cells, but more in type II and III cells (−31%) than in type I cells (−18%). Velocity of relaxation (+dL/dt) was also depressed more in type II and III cells (−38%) than in type I cells (−16%). Using fura 2 dye with intracellular Ca²⁺ transients, we demonstrated that in α-MHC^403/+ myocytes, the amplitude of the Ca²⁺ signal during contraction was unchanged but that the time required for decay of the signal to decrease 70% from its maximum was delayed significantly (WT = 159 ± 8 ms; α-MHC^403/+ = 217 ± 14 ms,P < 0.01). Sarco(endo)plasmic reticulum Ca²⁺-ATPase mRNA levels in α-MHC^403/+ and WT mice were similar. These data indicate that the altered cardiac dysfunction of α-MHC^403/+ myocytes is directly due to defective myocyte function rather than to secondary changes in global cardiac function and/or loading conditions.