The altered caffeine sensitivity of malignant hyperthermia-susceptible (MHS) muscle contracture is one basis of the diagnostic test for this syndrome. To determine whether the Arg615-to-Cys615 mutation of the porcine sarcoplasmic reticulum (SR) Ca2+ release channel is directly responsible for this altered caffeine sensitivity, the single-channel kinetics of purified MHS and normal pig Ca2+ release channels were examined. Initial studies demonstrated that decreasing the pH of the medium in either the cis- or trans-chamber decreased the Ca2+ release channel percent open time (Po). The half-inhibitory pH of MHS channels (6.86 +/- 0.04, n = 17) was significantly different from that of normal channels (7.08 +/- 0.07, n = 14). At pH 7.4, in either 7 or 0.12 microM Ca2+, MHS channel Po was not significantly different from that of normal channels over the range 0-10 mM caffeine. Although at pH 6.8 in 7 microM Ca2+ MHS channel Po was greater than that of normal channels over the range 0-20 mM caffeine, the difference could be eliminated by dividing each mean MHS Po by a scaling factor of 3.2. Thus the MHS Ca2+ release channel mutation does not appear to be directly responsible for the altered caffeine sensitivity of MHS pig muscle contracture. Rather, this altered caffeine sensitivity may result from an altered resting myoplasmic Ca2+ concentration or the altered pH and Ca2+ sensitivity of Ca2+ release channel Po of MHS muscle.