In principle, the force produced by cardiac muscle can be increased by generating a larger Ca²⁺ transient or be modifying the Ca²⁺ sensitivity of the myofilaments. We carried out experiments to identify the mechanism by which a novel positive inotropic compound with structural similarities to steroid hormones exerts its action on Ca²⁺ signaling. Two enantiomers were at our disposal, the active form (F90927) was known to increase the contractile force in Langendorff perfused hearts, whereas F90926 (inactive enantiomer) did not show an effect and served as a negative control. The whole-cell patch clamp technique was used with ventricular myocytes isolated from humanely killed guinea-pigs, in combination with UV laser-flash photolysis of caged Ca²⁺ (DMnitrophen) (DelPrincipe et al. 1999). Initially, I_CaL,I_Na and INCX were examined (DelPrincipe et al.2000, Lipp et al. 2002). The active enantiomer increased I_CaL about 2.5 times at a concentration of 10 µM, while I_Na and I_NCX remained unaffected. The inactive enantiomer did not change any of the currents investigated. Therefore, a more detailed study of I_CaL was carried out. The dose-response curve showed a biphasic increase of I_CaL with two Kd of ≈ 10 nM and ≈ 2 µM (n=3), respectively. The current-voltage relationship exhibited a leftward shift by 8.5 ± 0.5 mV (n=4, S.D.) in the presence of 10 µM of the active enantiomer. Next, we examined the voltage-dependence of activation and ^ICaL inactivation. These results revealed a shift of the activation curve by 20.4 mV (n=4) towards more negative potentials (at 10 µM), whereas the inactivation curve exhibited no shift. The inactive enantiomer did not affect the current-voltage relationship of I_CaL (n=3). We conclude that F90927 exerts its positive inotropic effect as an L-type Ca²⁺ channel agonist, by shifting the voltage-dependent activation towards more negative potentials. There appear to be two binding sites for F90927 on the channel or on regulatory proteins, exhibiting distinctly different affinities. In future studies we will identify the binding sites and molecular mechanisms underlying stimulation of the L-type Ca²⁺ channels by F90927.