The specific activity of d-glyceraldehyde-3-phosphate (G3P) dehydrogenase (phosphorylating) (GPDH, EC 1.2.1.12) found in skeletal muscle of induced hibernating jerboa (Jaculus orientalis) was 3–4 fold lower than in the euthermic animal. The comparative analysis of the soluble protein fraction of these tissues by SDS-PAGE and Western blotting showed a significant decrease in the intensity of a protein band of about 36 kDa, the GPDH subunit, in hibernating jerboa. After using the same purification procedure, the GPDH from muscle of hibernating jerboa exhibited lower values for both apparent optimal temperature and specific activity than the enzyme from the euthermic animal. Non-linear Arrhenius plots were obtained in both cases, but the E_a values calculated for the GPDH from hibernating tissue were higher. Although in both purified enzyme preparations three isoelectric GPDH isoforms, exhibiting pI values in the range 8.2–7.5, were resolved by chromatofocusing, clear differences were observed in these preparations concerning the relative contribution to the total enzymatic activity of the two main isoforms, named GPDH (pI values, 8.1–8.2) and GPDH II (pI values, 7.8–7.9). Thus, whereas GPDH I was the major isoform purified from euthermic muscle, accounting for more than 90% of the total activity, the amount of activity due to GPDH II reached up to 65% in preparations of hibernating jerboa. All isoforms exhibited similar native and subunit molecular masses and cross-reacted with an anti-GPDH antibody raised against the GPDH I. However, the two muscle GPDH isoforms prevailing under hibernating conditions exhibited a decreased catalytic efficiency when compared with the corresponding major isoforms purified from euthermic animals, as indicated by their different specific activities and kinetic parameters, i.e. relatively high K_m and low V_max values. Since the glycolytic flow has been found to be widely reduced in skeletal muscle of induced hibernating jerboa, the changes in the GPDH isoforms described in the present study could provide a molecular basis to explain some of the metabolic changes associated with mammalian hibernation.