Transverse tubule (TT) calcium transport and permeability were examined in the inherited skeletal muscle disorder malignant hyperthermia (MH). ATP-dependent calcium uptake by TT vesicles isolated from normal and MH-susceptible (MHS) pig muscle had a similar dependence on ionized Ca2+ concentration (K 1 2 for Ca2+ of 0.21 ± 0.04 and 0.25 ± 0.05 μm for MHS and normal TT, respectively), as well as a similar Vmax (20.9 ± 2.0 and 23.7 ± 4.5 nmol Ca/mg protein/min for MHS and normal TT, respectively). Furthermore, the stimulation of calcium uptake by either calmodulin or cAMP-dependent protein kinase was similar in normal and MHS TT. Halothane concentrations greater than 2 mm inhibited calcium uptake by either normal or MHS TT to a similar extent (IC50 = 8 mm). Dantrolene (10 μm), nitrendipine (1 μm), and Bay K 8644 (1 μm) had no significant effect on either the initial rates of calcium uptake or maximal calcium accumulation of either MHS or normal TT vesicles. However, in the absence of any added agents, maximum calcium accumulation by MHS TT was significantly less than by normal TT (90 ± 10 versus 130 ± 9 nmol Ca/mg protein after 15 min of uptake). This difference was not due to an increased permeability of MHS TT to calcium, nor was it due to a difference in the sarcoplasmic reticulum contamination (less than 5%) of the MHS and normal preparations. Although our results indicate there is no significant defect in MHS TT calcium regulation, the diminished maximum calcium accumulation by MHS TT may contribute to the abnormal sarcoplasmic calcium homeostasis in skeletal muscle during an MH Crisis.