The present study extends our previous work regarding new antifolates for Mycobacterium avium (MAC) dihydrofolate reductase (DHFR). The objectives of this study were to synthesize and test new derivatives in the general class of 2,4-diamino-5-methyl-5-deazapteridines in an effort to improve solubility and selectivity for the MAC DHFR, while maintaining lack of selectivity for the human DHFR. New 6-[2′, 5′-dialkoxyphenyl) methyl]-substituted DMDP analogs were synthesized as previously described. Three clinical isolates of MAC (NJ211, NJ3404, and NJ168) and M. tuberculosis H37Ra (MTB) were used to evaluate the new derivatives. A previously described colorimetric (alamarBlue®) microdilution broth assay was used to determine minimal inhibitory concentrations (MIC). Purified recombinant human (rDHFR), MAC rDHFR, and MTB rDHFR were used in a validated enzyme assay to obtain IC50 values and to determine selectivity ratios (SR) for the derivatives. For the MAC strains, the MICs ranged from ≤ 0.25 to > 16 μg/mL. The most active derivative against MAC was SRI-20920 which had MICs of 0.25, 0.25, and 8 μg/mL for the three strains, respectively. The most selective derivative was SRI-20730 with IC50s of 29 and 67,781 nM for MAC rDHFR and hDHFR, respectively, and a SR of 2,337. MICs for MTB ranged from 4 to >64 μg/mL and the SR, in general, ranged from 0.32 to 2.5. These results further substantiate the utility of this group of DMDP derivatives for selective activity against MAC. © 2006 Bentham Science Publishers Ltd.