N alpha-(4-Amino-4-deoxy-N10-methylpteroyl)-N epsilon-(iodoacetyl)-L-lysine (1) was synthesized as a potential active-site-directed irreversible inhibitor of dihydrofolate reductase (DHFR). In an ultraviolet spectrophotometric assay of dihydrofolate reduction of Lactobacillus casei DHFR, 1 and methotrexate (MTX, 4-amino-4-deoxy-N10-methylpteroyl-L-glutamic acid) had ID50 values of 4.5 and 6.2 nM. The corresponding ID50 values in a competitive radioligand binding assay against [3H]MTX were 31 and 16 nM. Thus, as reversible inhibitors of this enzyme over a short exposure time, 1 and MTX had comparable activity. On the other hand, when L. casei DHFR was incubated for up to 6 h with 0.1 or 1.0 microM 1, a progressive decrease in the ability of [3H]MTX to subsequently displace the drug was observed. When MTX itself was used at the same concentrations, the extent of displacement of [3H]MTX did not decrease with time. These results were consistent with rapid reversible binding of 1 to the enzyme, followed more slowly by covalent bond formation near the active site. The pH profile for this effect followed a curve with a sigmoidal shape. The apparent inflection point near pH 7.2 was consistent with alkylation of a histidine residue.