The role of cardiac troponin C (cTnC) as a target protein for the calcium sensitization by levosimendan, pimobendan, MCI-154 and EMD 53998 was evaluated using purified recombinant human cTnC. For determination of calcium- and magnesium-dependent binding of the compounds to cTnC a new type of cTnC-HPLAC column was used. Furthermore, dansylated cTnC was utilized to study the effect of the calcium sensitizing compounds on calcium-induced conformation of cTnC. Only levosimendan showed calcium-dependent and to a lesser extent magnesium-dependent retention in the cTnC column. The findings indicate that levosimendan binds both to the N-terminal and C-terminal domains of cTnC. In agreement with this, only levosimendan shifted the calcium-induced fluorescence curve of dansylated cTnC to the left. In the control experiments Ca50 and KCa2+ were calculated to be 2.73 microM and 4 x 10(5) M-1, respectively. Levosimendan at 3 microM decreased the value of Ca50 to 1.19 microM. In conclusion, it is suggested that the mechanism of calcium sensitizing effect of levosimendan, unlike that of the other calcium sensitizers, is based on calcium-dependent binding to the N-terminal domain of cTnC. This is proposed to amplify the trigger of contraction induced by cTnC in the cardiac muscle.