The conformational properties of the lipophilic antifolate trimetrexate (TMQ) were calculated and compared to the structurally-analogous prototypical antifolate methotrexate (MTX) using both empirical force-field and AM1 quantum mechanical methods. The conformational preferences of TMQ and MTX are diametrically opposed with respect to the bridge-system set of torsion angles tau 1, tau 2: TMQ prefers gauche, trans while MTX prefers approximately trans, gauche. These predictions are consistent with the observed crystal structures of TMQ (i.e., tau 1 = 79 degrees, tau 2 = 178 degrees) and of DHFR-bound MTX (i.e., tau 1 = -157 degrees, tau 2 = 57 degrees in L. casei). The crystal structure of MTX.4H2O deviates from this pattern with tau 1 closer to cis (i.e., 39 degrees) than the predicted trans, yet this near-cis conformation is driven by intermolecular hydrogen-bonding and electrostatic forces operative in the MTX crystal. As a consequence of these strong intermolecular forces, MTX incurs 1.8 kcal/mole in conformational-strain energy in its crystalline form. In contrast, TMQ experiences virtually no conformational strain in its crystalline form. This disparity is attributed to two distinctions between TMQ and MTX: (i) MTX crystallizes as a zwitterion while TMQ crystallizes as the free base, and (ii) the hydrophilic glutamate tail in MTX is replaced by three lipophilic trimethoxy groups in TMQ. The corresponding conformational-strain energy of DHFR-bound MTX is 2.0 kcal/mole while that of DHFR-bound TMQ is only 0.65 kcal/mole based on the assumption that the latter adopts the same bridge conformation as the former. This cost in conformational-strain energy for TMQ and MTX is paid at the expense of their respective free energies of binding of DHFR. Consequently, the present study offers the possibility of designing a new class of antifolates which are conformationally strain-free when bound to DHFR and thereby more effective as chemotherapeutic agents.