OBJECTIVE To analyse the use of standardized application of ryanodine for in vitro muscle contracture testing to define cut-off values separating malignant hyperthermia susceptible from malignant hyperthermia negative subjects. Furthermore, we compared the results of in vitro muscle-contracture tests following the halothane, caffeine and ryanodine challenges. METHODS In 113 subjects, halothane, caffeine and ryanodine muscle-contracture tests were performed according to the protocol of the European Malignant Hyperthermia Group. RESULTS Malignant hyperthermia susceptible subjects (n = 77) had significantly shorter onset times in the ryanodine in vitro muscle-contracture test (1 micromol ryanodine) compared with malignant hyperthermia negative subjects (n = 36), median 4.8 vs. 20.1 min, respectively, without any influence of age or gender. The best cut-off value was 10 min (sensitivity 0.78 and specificity 0.94, respectively). Shorter cut-off values had greater specificity, but lower sensitivity. Groups could not be separated without an overlap. In susceptible subjects, we found a correlation between onset time and threshold concentrations for halothane and caffeine (p = 0.47 and 0.52, respectively). In addition, muscle bundles with high susceptibility to halothane and caffeine also showed high susceptibility to ryanodine. CONCLUSIONS The ryanodine in vitro muscle-contracture test confirmed the malignant hyperthermia status that was determined using the halothane and caffeine in vitro muscle-contracture tests. Due to an overlap between the two groups, discrimination ability was not always perfect and short cut-off values with higher specificity had reduced sensitivity and vice versa. The correlation of contractures following the halothane, caffeine and ryanodine challenges points towards a similar individual pharmacogenetic effect rather than a specific, different pharmacological action between the three agents.