We investigated the enzyme kinetic and antifolate inhibitory properties of human dihydrofolate reductase enzyme with mutations at position 22. Leu-22 was changed to isoleucine, methionine, phenylalanine, and tyrosine to generate the various mutant enzymes. The overall catalytic efficiency (kcat/Km) for methionine and phenylalanine mutants was reduced approximately 3-fold and >6-fold for isoleucine and tyrosine mutants. An arginine mutant (L22R) was also expressed but had a dramatically reduced catalytic potential (kcat>250-fold lower than wild-type) and therefore was not studied in detail. The Ki for antifolates, methotrexate, aminopterin, and trimetrexate are more dramatically affected (increased) than the Km for dihydrofolate, particularly for phenylalanine and tyrosine mutants. One remarkable feature is that the phenylalanine mutant is as potently inhibited by piritrexim as is the wild-type human enzyme, although the Ki values for methotrexate and aminopterin were increased 88- and 118-fold, respectively. This is likely related to different positioning of the methoxyphenyl side chain of piritrexim relative to the side chains of other compounds tested. A Chinese hamster cell line harboring the L22F mutant also demonstrated an increased sensitivity of piritrexim relative to antifolates.