Negative inotropic agents may differentially modulate indices of cytosolic [Ca²⁺]-left ventricular pressure (LVP) relationships when given before and after ischemia. We measured and calculated [Ca²⁺], LVP, velocity ratios [d[Ca²⁺]/dt_max]/[dLVP/dt_max] (VR_max), [d[Ca²⁺]/dt_min]/[dLVP/dt_min] (VR_min), and area ratio (AR, area[Ca²⁺]/area LVP per beat) before and after global ischemia in guinea pig isolated hearts. Ca²⁺ transients were recorded by indo 1-AM fluorescence via a fiber optic probe placed at the LV free wall. [Ca²⁺]-LVP loops were acquired by plotting LVP as a function of [Ca²⁺] at multiple time points during the cardiac cycle. Hearts were perfused with bimakalim, 2,3-butanedione monoxime (BDM) nifedipine, or lidocaine before and after 30 min ischemia. Before ischemia each drug depressed LVP, but only nifedipine decreased both LVP and [Ca²⁺] with a down- and leftward shift of the Ca²⁺-LVP loop. After ischemia each drug depressed LVP and [Ca²⁺] with a down and leftward shift of the Ca²⁺-LVP loop. Each drug except BDM decreased dCa²⁺/dt_max; nifedipine decreased dCa²⁺/dt_min, whereas lidocaine increased it and bimakalim and BDM had no effect on dCa²⁺/dt_min. Each drug except bimakalim increased VR_max and VR_min before ischemia; after ischemia only BDM and nifedipine increased VR_max and VR_min. Before and after ischemia, BDM and nifedipine increased AR, whereas lidocaine and bimakalim had no effect. At 30 min reperfusion, control hearts exhibited marked Ca²⁺ overload and depressed LVP. In each drug pre-treated group Ca²⁺ overload was reduced on reperfusion but only the group pre-treated with nifedipine exhibited both higher LVP and lower [Ca²⁺]. These results show that negative inotropic drugs are less capable of reducing [Ca²⁺] after ischemia so that there is a relatively larger Ca²⁺ expenditure for contraction/relaxation after ischemia than before ischemia. Moreover, the differential effects of pre-treatment with negative inotropic drugs on Ca²⁺-LVP relationships after ischemia suggest that these drugs, especially nifedipine, can elicit cardiac preconditioning.