Twelve novel 2,4-diamino-5-(4'-benzylamino)- and 2,4-diamino-5[4'-(N-methylbenzylamino)-3'-nitrophenyl]-6-ethylp yrimidines bearing 4-substituents on the benzylamino or N-methylbenzylamino aryl ring were synthesized and evaluated as nonclassical inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase (DHFR). Compounds were prepared by reaction of 2,4-diamino-5-(4'-chloro-3'-nitrophenyl)- (8) or 2,4-diamino-5-(4'-fluoro-3'-nitrophenyl)-6-ethylpyrimidine (15) with the appropriate 4-substituted (CO2H, CO2Me, SO2NH2, dioxolan-2-yl, CHO, dimethyloxazolin-2-yl) benzylamine or N-methylbenzylamine derivative. Compounds 25-29 were synthesized from 2,4-diamino-5-{4'-[N-(4"-carboxybenzyl)amino]-3'-nitrophenyl}-6- ethylpyrimidine (10) and the corresponding amine (NH3, MeNH2, Me2NH, piperidine, diethyl L-glutamate) via isobutyl mixed anhydride coupling; hydrolysis of the diethyl L-glutamate 29 afforded the L-glutamate analogue 30. The compounds exhibited potent inhibitory activity against T. gondii (IC50 values 0.0018-0.14 microM) and rat liver (IC50 values 0.0029-0.27 microM) DHFR, with a 4-substituent invariably enhancing binding to both enzymes relative to the unsubstituted benzoprim (5) or methylbenzoprim (6). Modest selectivity for T. gondii enzyme was observed with several analogues, whereas all of the compounds were relatively weak inhibitors of P. carinii DHFR and exhibited no selectivity. Selected analogues were evaluated for in vivo antitumor activity against the methotrexate-resistant M5076 murine reticulosarcoma, with 2,4-diamino-5-{4'-[N-[4"-(N"-methylcarbamoyl)benzyl]-N- methylamino]-3'-nitrophenyl}-6-ethylpyrimidine (14) (Ki for rat liver DHFR = 0.00035 +/- 0.00029 nM) combining significant antitumor activity with minimal toxicity.