The subtilisin-like serine proteinases, VPR, from a psychrotrophic Vibrio species and aqualysin I (AQUI) from the thermophile Thermus aquaticus, are structural homologues, but differ significantly with respect to stability and catalytic properties. It has been postulated that the higher catalytic activity of cold adapted enzymes when compared to homologues from thermophiles, reflects their higher molecular flexibility. To assess a potential difference in molecular flexibility between the two homologous proteinases, we have measured their Trp fluorescence quenching by acrylamide at different temperatures. We also investigated protein dynamics of VPR and AQUI at an atomic level by molecular dynamics simulations. VPR contains four Trp residues, three of which are at corresponding sites in the structure of AQUI. To aid in the comparison, a Tyr at the fourth corresponding site in AQUI was mutated to Trp (Y191W). A lower quenching effect of acrylamide on the intrinsic fluorescence of the thermophilic AQUI_Y191W was observed at all temperatures measured (10-55°C), suggesting that it possesses a more rigid structure than VPR. The MD analysis (Cα rmsf profiles) showed that even though VPR and AQUI have similar flexibility profiles, the cold adapted VPR displays higher flexibility in most regions of the protein structure. Some of these regions contain or are in proximity to some of the Trp residues (Trp6, Trp114 and Trp208) in the proteins. Thus, we observe an overall agreement between the fluorescence quenching data and the flexibility profiles obtained from the MD simulations to different flexibilities of specific regions in the proteins.