The sarcoplasmic reticulum (SR) Ca2+ release channel (RyR1) from malignant hyperthermia-susceptible (MHS) porcine skeletal muscle has a decreased sensitivity to inhibition by Mg2+. This diminished Mg2+ inhibition has been attributed to a lower Mg2+ affinity of the inhibition (I) site. To determine whether alterations in the Ca2+ and Mg2+ affinity of the activation (A) site contribute to the altered Mg2+ inhibition, we estimated the Ca2+ and Mg2+ affinities of the A- and I-sites of normal and MHS RyR1. Compared with normal SR, MHS SR required less Ca2+ to half-maximally activate [3H]ryanodine binding (KA,Ca: MHS = 0.17 ± 0.01 μM; normal = 0.29 ± 0.02 μM) and more Ca2+ to half-maximally inhibit ryanodine binding (KI,Ca: MHS = 519.3 ± 48.7 μM; normal = 293.3 ± 24.2 μM). The apparent Mg2+ affinity constants of the MHS RyR1 A- and I-sites were approximately twice those of the A- and I-sites of the normal RyR1 (KA,Mg: MHS = 44.36 ± 4.54 μM; normal = 21.59 ± 1.66 μM; KI,Mg: MHS = 660.8 ± 53.0 μM; normal = 299.2 ± 24.5 μM). Thus, the reduced Mg2+ inhibition of the MHS RyR1 compared with the normal RyR1 is due to both an enhanced selectivity of the MHS RyR1 A-site for Ca2+ over Mg2+ and a reduced Mg2+ affinity of the I-site.