In an attempt to compare the results of physiologic contracture tests for malignant hyperthermia (MH) susceptibility between laboratories, a testing protocol was agreed upon by several investigators. This protocol was followed in order to determine which of several procedures is best able to identify MH susceptibility. Muscle biopsies were obtained from 12 normal control patients, 6 survivors of an MH episode, 24 patients who experienced succinylcholine-induced masseter muscle rigidity, 15 patients with a family history of MH, and 12 patients who had adverse reactions to anesthesia (not clearly MH). In all cases muscle strips were stimulated electrically and the contracture responses of different strips to halothane alone, caffeine alone, and caffeine in the presence of 1 % halothane were evaluated. In addition to the contracture response the caffeine-specific concentration was calculated (concentration of caffeine needed to produce a 1-g contracture). The procedures best able to discriminate MH positives from controls employed halothane alone or caffeine alone. Muscle contracture of 0.772 ± 0.585 g (mean ± SEM) was observed in biopsies from known MH susceptibles in response to 2 mM caffeine; the response of control muscle was significantly different (0.17 ± 0.209 g). Halothane contractures of 1.425 ± 1.098 g were recorded from muscle from known MH patients while contractures of 0.023 ± 0.169 g were found in control patients (significantly different). Using a caffeine-specific concentration of 2.2 mM, 10 of 15 normal people were misclassified as MH positive: using a halothane-caffeine-specific concentration of 0.7 mM, 4 of 15 normal people were misclassified as MH positive. Twelve of 21 patients presenting with succinylcholine-induced masseter muscle rigidity were MH positive, 50% of these had a family history of MH that was positive, and 1 of 12 with an “adverse” reaction to anesthesia was MH positive. The present results confirm the clinical utility of in vitro muscle testing in the diagnosis of MH and suggest other areas of usefulness including evaluation of the clinical significance of isolated adverse reactions to anesthesia and counseling of families having members known to be MH susceptible.