In vitro muscle contracture tests for malignant hyperthermia screening are routinely performed using standardized protocols. In the present study on-line monitoring of halothane concentrations in the gas phase was demonstrated to be an improved test standard. The kinetics of halothane concentration and their effect on in vitro muscle contracture tests were evaluated in two test baths, I and II, which contained 3 and 18 ml Krebs-Ringer solution, respectively. The equilibration kinetics for halothane was significantly faster in bath I (t1/2 = 8.2 s) compared with bath II (t1/2 = 25.6 s). Twenty-one pairs of muscle bundles from 21 potentially malignant hyperthermia susceptible patients were investigated, each test bath receiving one bundle from each pair. The variance of muscle contractures was significantly increased in test bath I compared with test bath II. However, there was no influence on malignant hyperthermia diagnosis, suggesting that, within the ranges of t1/2 = 8.2 s-25.6 s, the test bath volumes need not be standardized.