3,4,5-Triethylacetophenone was synthesized in 60% yield by a Friedel-Crafts reaction from 4-ethylacetophenone and converted to 2,4-diamino-5-(3,4,5-triethylbenzyl)pyrimidine (2), a trimethoprim (1) isostere, by standard techniques. This compound is more lipophilic than 1 by three log units (log P, octanol/water). Compound 2 was approximately equipotent with 1 in inhibiting Escherichia coli dihydrofolate reductase (DHFR), 2-fold more potent against P. berghei and N. gonorrhoeae DHFR, and 10 and 25 times better an inhibitor of rat and chicken liver DHFR, respectively. Although the 3,4-dimethoxy analogue 19 was 10-fold less inhibitory to E. coli DHFR than 1, it was 3-4 times more potent on the vertebrate isozymes, whereas the diethyl congener 10 followed 19 in its E. coli DHFR binding but was less active on rat and chicken DHFR. Therefore, a significant portion of the selectivity of 1 for bacterial, as opposed to vertebrate, DHFR, involves the methoxy functions. An analysis of the X-ray data on 1 and 2 complexed with chicken DHFR, coupled with kinetic data, led to the conclusion that the difference in binding energies of the methoxy and ethyl compounds probably involve desolvation factors, as well as direct energies of interaction with protein atoms. Thus, one cannot invoke lipophilicity or shape alone in explaining the relationship in properties of 1 and 2.