Propargyl-linked antifolates that target dihydrofolate reductase are potent inhibitors of several species of pathogenic bacteria and fungi. This novel class of antifolates possesses a relatively uncommon acetylenic linker designed to span a narrow passage in the enzyme active site and join two larger functional domains. Because the use of alkyne functionality in drug molecules is limited, it was important to evaluate some key physicochemical properties of these molecules and specifically to assess the overall stability of the acetylene. Herein, we report studies on four compounds from our lead series that vary specifically in the environment of the alkyne. We show that the compounds are soluble, chemically stable in water, as well as simulated gastric and intestinal fluids with half-lives of approximately 30 min after incubation with mouse liver microsomes. Their primary in vitro route of metabolism involves oxidative transformations of pendant functionality with little direct alteration of the alkyne. Identification of several major metabolites indicated the formation of N-oxides; the rate of formation of these oxides was highly influenced by branching substitutions around the propargyl linker. On the basis of the lessons of these metabolic studies, a more advanced inhibitor was designed, synthesized, and shown to have increased (t(1/2) = 65 min) metabolic stability while maintaining potent enzyme inhibition.