The F-actin disrupter cytochalasin D depresses cardiac contractility, an effect previously ascribed to the interaction of cytochalasin D with cytoskeletal actin. We have investigated the possibility that this negative inotropic effect is due to the interaction of cytochalasin D with sarcomeric actin of the thin filament. Confocal images of Triton X-100-skinned myocytes incubated with a fluorescent conjugate of cytochalasin D revealed a longitudinally striated pattern of binding, consistent with a myofibrillar rather than cytoskeletal structure.Tension-pCa relationships were determined at sarcomere lengths (SLs) of 2.0 and 2.3 [mu]m following 2 min incubation with 1 [mu]M cytochalasin D. Cytochalasin D significantly reduced the pCa for half-maximal activation (pCa50) at both SLs. The shift in pCa50 was significantly greater at a SL of 2.3 [mu]m compared with that at a SL of 2.0 [mu]m. Cytochalasin D had no effect on the Hill co-efficient at either SL. Cytochalasin D significantly reduced the maximum tension at both SLs. We suggest that the length-dependent decrease in myofilament Ca2+ sensitivity in response to cytochalasin D is due to a decrease in the affinity of troponin C for Ca2+. Cytochalasin D has been used for many years as the agent of choice for disruption of cytoskeletal actin. However, we have demonstrated for the first time an interaction of cytochalasin D with sarcomeric actin of the thin filament, which can account for the effects of cytochalasin D on cardiac contractility.