Enhanced sensitivity to caffeine is part of the standard tests for susceptibility to malignant hyperthermia (MH) in humans and pigs. The caffeine-sensitivity of skeletal muscle contraction and Ca²⁺ release from the sarcoplasmic reticulum is enhanced, but surprisingly, caffeine sensitivity of purified porcine ryanodine receptor Ca²⁺ release channels (RyRs) is not affected by the MH mutation (Arg⁶¹⁵Cys). In contrast, we show here that native malignant hyperthermic pig RyRs (incorporated into lipid bilayers with RyR-associated lipids and proteins), were activated by caffeine at 100-1000-fold lower concentrations than native normal pig RyRs. In addition, the results show that the mutant ryanodine receptor channels were less sensitive to high affinity activation by a peptide (C_S) which corresponds to a part of the II-III loop of the skeletal dihydropyridine receptor. Furthermore, sub-activating concentrations of peptide C_S enhanced the response of normal pig and rabbit RyRs to caffeine. In contrast, the caffeine-sensitvity of MH RyRs was not enhanced by the peptide. These novel results showed that in MH-susceptible pig muscles (a) the caffeine-sensitivity of native RyRs was enhanced, (b) the sensitivity of RyRs to a skeletal II-III loop peptide was depressed and (c) an interaction between the caffeine and peptide C_S activation mechanisms seen in normal RyRs was lost.