Quantitative structure-activity relationships for the inhibition of Escherichia coli (MB 1428) dihydrofolate reductase (DHFR) by 61 5-(substituted benzyl)-2,4-diaminopyrimidines are reported and analyzed. The 61 compounds include 17 congeners whose activities have not been previously reported, five of which have a 5'-substituent larger than a methoxy group. The correlation equations indicated that the molar refractivity (MR) values of the 5'-substituent, just as with the 3'- and 4'-substituents, contributed maximally at the value of 0.79 with no increment of binding for compounds with MR larger than 0.79 (which corresponds to a 5'-methoxy substitution). This experimental result is in agreement with the crystal structure of the Escherichia coli DHFR-trimethoprim complex, which shows a reasonably large trimethoprim-binding site. The inhibition of E. coli (MB 1428) DHFR by nine of the 17 new benzylpyrimidines is at lower concentrations than for trimethoprim. However, all 17 are much less potent than trimethoprim in inhibition of growth of E. coli (1515).