Although cardiac steroids (CS) have long been used to treat cardiac insufficiency, the mechanism(s) of action of these agents remain open to question. While many results indicate that inhibition of Na⁺,K⁺-ATPase underlies both the therapeutic and toxic actions of CS, other studies suggest that actions on the SR membrane system may be important. We used two experimental approaches and measurements of left ventricular diastolic pressure (LVDP) in isolated guinea pig hearts to test whether CS had an intracellular site of action. In the first approach, we compared the inotropic effects of a hydrophilic CS, ouabain, and a hydrophobic CS, digitoxin, after the activity of the Na⁺ pump was reduced by perfusing hearts with solutions maintained at 5°C. Under these conditions, exposure of hearts to 1 μM ouabain for 60 min did not increase LVDP above control levels. In contrast, an equi-effective concentration of digitoxin (0.3 μM) increased LVDP by 40 ± 8.5% (p<0.01) over pre-drug control levels. In the second experimental approach, we compared the inotropic effects of ouabain and digitoxin in the presence of rapid-cooling contractures (RCC), which result in the release of SR Ca²⁺. Hearts were perfused with Tyrode solution or Tyrode solution containing either digitoxin (0.3 μM) or ouabain (1 μM) for 180 sec, rapidly cooled and the RCC responses were analyzed. Compared to RCC elicited in Tyrode solution alone, or in Tyrode solution containing ouabain, RCC in the presence of digitoxin reached peak amplitudes more rapidly, but elicited reduced peak amplitude values. Based on these findings, we suggest that: 1) the ability of the hydrophobic CS, digitoxin, but not the hydrophilic CS, ouabain, to produce a positive inotropic effect at 5°C, when the activity of the Na⁺ pump is markedly reduced, is consistent with a mechanism other than Na⁺ pump inhibition and involves an intracellular location; and 2) the diminished RCC observed in the presence of the hydrophobic CS, digitoxin, indicate that this alternative mechanism may involve effects on the SR Ca²⁺ release channel.