The effects of 2,3-butanedione monoxime (BDM) and 5-[1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydro-6-quinolyl]-6-me thy l-3,6-dihydro-2H-1,3,4-thiadiazin-2-one (EMD 53998) on cardiac muscle were studied in skinned muscle fibres from the right ventricle of the porcine heart. BDM decreases the Ca2+ sensitivity (pCa50 for 50% activation) and it exerts a dose-dependent inhibitory effect on force in troponin I (TnI)-depleted (unregulated) cardiac skinned muscle fibres (IC50 approximately 20 mM) thereby mimicking the effect of the TnI inhibitory peptide (cTnI 137-148, corresponding to the cardiac TnI inhibitory region) and that of inorganic phosphate (Pi). This inhibitory action can be antagonized by the calcium-sensitizing cardiotonic thiadiazinone derivative EMD 53998 that increases the IC50 to about 30 mM. In skinned fibres, BDM (10 mM) also increased the ratio of ATPase activity to isometric force (tension cost), whereas EMD 53998 (20 mu M) decreased it. We propose that BDM antagonizes EMD 53998 because both compounds affect the Pi release step of the crossbridge cycle in an antagonistic manner.