Analyses of temperature-dependent kinetic parameters in enzymes extracted from tissues of ectothermic animals are usually carried out within the range of physiological temperatures (0-40°C). However, multisample spectrophotometers (so-called microplate readers) with efficient wide-range temperature control (including cooling) have previously been unavailable. This limits the statistical quality of the measurements. A temperature-controlled microplate was designed for a 96-well microplate reader to overcome this limitation. This so-called T-microplate is able to control assay temperature between the freezing point of a liquid sample and 60°C with high stability and accuracy in any data acquisition mode. At 4°C the accuracy of the temperature control was ±0.1°C and temperature homogeneity across the microplate was ±0.3°C. As examples, analyses of the temperature dependence of Michaelis-Menten (K′PYRm) and substrate inhibition (KsiPYR) constants for pyruvate, of the maximal rate of reaction (V′max), of the apparent Arrhenius activation energy (EA), and of the Gibbs free-energy change (ΔG‡) of lactate dehydrogenases from muscle of Atlantic cod Gadus morhua acclimated to 4°C are described. The large dataset obtained allowed evaluation of a new mechanism of metabolic compensation in response to seasonal temperature change. © 2004 Elsevier Inc. All rights reserved.