We report here the conformational preference and prolyl cis-trans isomerization of 4(R)-substituted proline dipeptides, N-acetyl-N'-methyl amides of 4(R)-hydroxy-L-proline and 4(R)-fluoro-L-Proline (Ac-Hyp-NHMe and Ac-Flp-NHMe, respectively), studied at the HF/6-31+G(d),B3LYP/6-31+G(d), and B3LYP/6-311++G(d,p) levels of theory. The 4(R)-substitution by electron-withdrawing groups did not result in significant changes in backbone torsion angles as well as endocyclic torsion angles of the prolyl ring. However, the small changes in backbone torsion angles phi and psi and the decrease of bond lengths r(C-beta-C-gamma) or r(C-gamma-C-delta) appear to induce the increase of the relative stability of the trails up-puckered conformation and to alter the relative stabilities of transition states for prolyl cis-trans isomerization. Solvation free energies of local minima and transition states in chloroform and water were calculated using the conductor-like polarizable continuum model at the HF/6-31+G(d) level of theory. The Population of trans up-puckered conformations increases in the order Ac-Pro-NHMe < Ac-Hyp-NHMe < Ac-Flp-NHMe in chloroform and water. The increase in population for trails Up-pUckered conformations in solution is attributed to the increase in population for the polyproline-II-like conformations with up puckering. The barriers Delta G(ct)(double dagger) to prolyl cis-to-trans isomerization for Ac-Hyp-NHMe and Ac-Flp-NHMe increase as the solvent polarity increases, as seen for Ac-Pro-NHMe. In particular, it was identified that the cis-trans isomerization proceeds through the clockwise rotation about the prolyl peptide bond for Ac-Hyp-NHMe and Ac-Flp-NHMe in chloroform and water, as seen for Ac-Pro-NHMe.