A series of 2,4-diamino-5-aryl-6-ethylpyrimidines embracing basic substituents in the 5-aryl ring was synthesized and evaluated for inhibitory activity against rat liver dihydrofolate reductase (DHFR). Maximal enzyme inhibition was observed for compounds bearing a benzylamino (19) or N-alkylbenzylamino substituent (29 and 30) in the 4-position of the phenyl ring and a nitro group in the 3-position, the corresponding 3-amino, 3-azido, or unsubstituted analogues proving only weakly active or inactive as DHFR inhibitors. Selected compounds were also screened in vivo against a methotrexate-resistant tumor, the M5076 murine reticulosarcoma, and antitumor activity in general paralleled activity against DHFR, the (3,4-dichlorobenzyl)amino analogue 26 proving the least toxic compound to exhibit significant antitumor activity. The X-ray crystal structure of the ethanesulfonic acid salt of the N-methylbenzylamino compound 29 has been determined to facilitate future molecular modeling studies in this new series of DHFR inhibitors.