Isolated guinea pig papillary muscles were subjected to an in vitro model of ischemia, consisting of superfusion arrest and immersion in paraffin oil, which results in restriction of substrate supply and metabolite washout. Intracellular pH (pHi) and surface pH (pHs) were measured with glass microelectrodes. Contractile force declined to 82% of the pre-“ischemic” value after 2 min and to 37% of the control value after 10 min. In addition, a shortening of the time to peak and duration of contraction was noted. The rate of force development decreased later than the rate of relaxation. After 10 min, pHi was acidified on average 0.08 pH unit, which is about one-third of the measured pHs change. Tripling the ischemic pHi change by reduction of the intracellular buffering power only slightly increased the rate of tension decline. Experimental pHi changes of similar magnitude, induced during normal superfusion, had a smaller effect on contractile force and failed to reproduce the characteristic changes in time course of the contraction. It is concluded that, in our condition of simulated ischemia, the intracellular acidification cannot account fully for the rapid decline in contractility.