The capacities of subtilisin Carlsberg (SC) and alpha-chymotrypsin (CT), which are representative of synthetically useful serine proteases, to discriminate between R-and S-configurations of stereocenters remote from the catalytic site have been further explored using chiral naphthyl aldehyde transition state analog inhibitors as probes. The inhibitors evaluated were (3R)- and (3S)-3-(1-naphthyl and 2-naphthyl)butanal and (4R)- and (4S)-4-(1-naphthyl and 2-naphthyl)pentanal, for which the methyl groups at the C-3 and C-4 stereocenters, respectively, are significantly removed from the aldehyde functionality that interactswith the catalytic serine residue. Each aldehyde was a competitive inhibitor for both enzymes, with CT being significantly more powerfully inhibited than SC. While only low levels of stereoselectivity were observed with SC, significant stereocenter discrimination was manifest for CT within this series of inhibitors with, encouragingly, the similar degrees of stereoselectivity (up to 3.9-fold) being observed between the enantiomers of the naphthylaldehyde inhibitors bearing both C-3 and C-4 remote methyl substituents. Furthermore, CT consistently favored the S-enantiomers within this series of inhibitor structures, which represents a reversal of the stereoselectivity observed previously for inhibition by the analogous phenyl substituted aldehydes. Molecular mechanics and molecular dynamics calculations were performed to identify the binding and orientation differences responsible for the R-and S-enantiomer binding discriminations observed.