Secophalloidin (SPH) is known to activate skinned cardiac muscle in the absence of Ca2+. We hypothesized that SPH-induced changes in cross-bridge properties underlie muscle activation. We found that force responsiveness to orthovanadate was drastically reduced in SPH activated muscles compared to Ca2+-activated contraction. Moreover, SPH caused similar to30% increase in Ca2+-independent force in muscles where Ca2+ sensitivity was totally destroyed by troponin I extraction with 10 mM vanadate. Thus, SPH and Ca2+ activation differ in both properties of the cross-bridge cycle and protein requirements for thin filament regulation. In addition, we tested the relationship between the activating effects SPH and EMD 57033, a Ca2+ sensitizer that increases resting force in cardiac muscle. After maximal activation by either SPH or EMD 57033, the other compound was found to further increase force, indicating that SPH activates muscle via a novel mechanism. (C) 2003 Elsevier Science (USA). All rights reserved.