The free-swimming ciliate Paramecium avoids warm regions. We have developed a quantitative assay of thermal avoidance, based on the interference between thermal avoidance and the normal tendency to swim upward (negative geotaxis). Paramecium tetraurelia swimming in a Tris/Ca2+ solution avoided a region warmer than about 40 "C. Several factors influenced the strength of the response, including the concentrations of monovalent cations and Ca2+, the temperature of the test region, and the temperature at which the animals had been cultured. At 1 mM-Ca2+, increasing Na+ concentration enhanced avoidance of a 40 "C region, and at a constant Na+ concentration (0.5 mM), avoidance improved with decreased Ca2+. When the ratio [Na+]/[Ca2+]i was held constant, varying Na+ and Ca2+ concentrations did not affect thermal avoidance. Other monovalent cations (Cs-', Li+, Rb+) and hydroxylamine also enhanced thermal avoidance, and K+ was somewhat less effective than these. The strength of the avoidance response increased with increasing test temperature in the range of 37 to 42 "C for cells grown at 28 "C. Cells grown at 15 "C had a lower threshold for thermal avoidance, and those grown at 35 "C showed no avoidance at 40 "C and only poor avoidance at higher temperatures. Cells cultured at 15 or 35 "C and then shifted to 28 "C acquired the thermal behaviour typical of cells cultured at 28 "C. Behavioural mutants with defective Ca2+ channels (Pawns) are incapable of reversing their swimming direction and showed little or no thermal avoidance. We suggest that thermal avoidance is triggered by thermotropic phase transitions in the lipids of the excitable membrane of Paramecium tetraurelia.