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To gain insight into the molecular determinants of thermoadaptation within the family of archaeal glyceraldehyde-3-phosphate dehydrogenases (GAPDH), a homology-based 3-D model of the mesophilic GAPDH from Methanobacterium bryantii was built and compared with the crystal structure of the thermophilic GAPDH from Methanothermus fervidus. The homotetrameric model of the holoenzyme was initially assembled from identical subunits completed with NADP molecules. The structure was then refined by energy minimization and simulated-annealing procedures. PROCHECK and the 3-D profile method were used to appraise the model reliability. Striking molecular features underlying the difference in stability between the enzymes were deduced from their structural comparison. First, both the increase in hydrophobic contacts and the decrease in accessibility to the protein core were shown to discriminate in favor of the thermophilic enzyme. Besides, but to a lesser degree, the number of ion pairs involved in cooperative clusters appeared to correlate with thermostability. Finally, the decreased stability of the mesophilic enzyme was also predicted to proceed from both the lack of charge-dipole interactions within alpha-helices and the enhanced entropy of unfolding due to an increase in chain flexibility. Thus, archaeal GAPDHs appear to be governed by thermoadaptation rules that differ in some aspects from those previously observed within their eubacterial counterparts., (Copyright 2002 Wiley Periodicals, Inc. Biopolymers 65: 263-273, 2002)