The crystal structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor, South China sea lobster, was determined and refined at 2 A resolution to an R factor of 17.1% and reasonable stereochemistry. The structure refinement has not altered the overall structure of GAPDH from this lobster species. However, some local changes in conformation and the inclusion of ordered solvent model have resulted in a substantial improvement in the accuracy of the structure. Structure analysis reveals that the two subunits including NAD+ in the asymmetric unit are remarkably similar. The thermal differences between the two subunits found in some regions of the NAD+-binding domain may originate from different crystallographic environments rather than from an inherent molecular asymmetry. In this structure, the side chain of Arg194 does not point toward the active site but forms an ion pair with Asp293 from a neighboring subunit. Structural comparisons with other GAPDH's of known structure reveal that obvious contrast exists between mesophilic and thermophilic GAPDH mainly in the catalytic domain with significant conformational differences in the S-loop, beta7-strand and loop 120-125; the P-axis interface is more conserved than the R- and Q-axis interfaces and the catalytic domain is more conserved than the NAD+-binding domain. Some possible factors affecting the thermostability of this enzyme are tentatively analyzed by comparison with the highly refined structures of thermophilic enzymes.