Force-velocity relations were obtained from single cardiac myocytes isolated by enzymatic digestion of rat myocardium and permeabilized with the pore-forming staphylococcal toxin α-hemolysin. Single cardiac myocytes were attached to a force transducer and piezoelectric translator and viewed with an inverted microscope to allow periodic monitoring of sarcomere length during experiments. Permeabilized cells were activated by immersion in a bath of known [Ca2+]. We report that the Ca2+ sensitivity of cells obtained by enzymatic digestion and permeabilized using α-hemolysin is similar to that reported previously for mechanically disrupted ventricular myocardium; however, the tension-pCa relation is less steep in the new preparation. During isotonic measurements, force was clamped to various loads using a rapid-response servo system. All recordings of shortening under load were distinctly curvilinear, and analysis of data involved fitting each shortening recording with a single exponential curve and calculating the value of the slope at the initial time of the load clamp. In addition, the presence of significant resting force at initial sarcomere lengths in these cells required that the possibility of alteration of velocity due to the presence of resting force be addressed. The maximum shortening velocity in fully Ca2+-activated single ventricular myocytes studied by this method was 2.83 muscle lengths per second on average. The basis for curvilinear shortening is postulated to be multifactorial in cardiac muscle, involving a combination of shortening inactivation and one or more passive elasticities that resist stretch or compression depending on sarcomere length. Shortening velocity shows a dependence on myosin isoform content when cells from a single heart are compared; however, this relation does not hold when cells from different hearts are compared. The behavior of single α-hemolysin-permeabilized myocyte shortening under loaded conditions at lower levels of Ca2+ is also described. During submaximal Ca2+ activation, initial shortening velocities are faster than those observed in maximally activated cells. This may be due to contributions of high passive force to increase shortening velocity under conditions of low active force generation, when passive force in the cell is a greater proportion of the total force and there are fewer bound crossbridges.