Phalloidin binds very tightly and specifically to actin and brings about a marked stabilization of the F-actin filament. In this study the effects of phalloidin on force generation and Ca²⁺ sensitivity of skinned bovine ventricular muscle were investigated. At all free Ca²⁺ concentrations addition of phalloidin to activated fibers caused an enhancement of active force. At full Ca²⁺ activation the force increase was about 6% and the relative force enhancement became greater as the Ca²⁺ concentration was decreased. Force-pCa plots obtained with fibers pre-treated with phalloidin showed that phalloidin produced an approximately 0.2 pCa unit increase in Ca²⁺ sensitivity without significant changes in cooperativity of activation. These results suggest that interactions between G-actin subunits may play an important role in cardiac force development.