The chemokine receptors CXCR1/2 are involved in a variety of inflammatory diseases, including chronic obstructive pulmonary disease. Several classes of allosteric small-molecule CXCR1/2 antagonists have been developed. The data presented here describe the cellular pharmacology of the acid and ester forms of the nicotinamide glycolate pharmacophore, a potent antagonist of CXCR2 signaling by the chemokines CXCL1 and CXCL8. Ester forms of the nicotinamide glycolate antagonized CXCL1-stimulated chemotaxis (IC(50) = 42 nM) and calcium flux (IC(50) = 48 nM) in human neutrophils, but they were inactive in cell-free assays of (125)I-CXCL8/CXCR2 binding and CXCL1-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) exchange. Acid forms of the nicotinamide glycolate were inactive in whole-cell assays of chemotaxis and calcium flux, but they inhibited (125)I-CXCL8/CXCR2 binding and CXCL1-stimulated [(35)S]GTPgammaS exchange. The (3)H ester was internalized by neutrophils and rapidly converted to the (3)H acid in a concentrative process. The (3)H acid was not internalized by neutrophils but was sufficient alone to inhibit CXCL1-stimulated calcium flux in neutrophils that were permeabilized by electroporation to permit its direct access to the cell interior. Neutrophil efflux of the acid was probenecid-sensitive, consistent with an organic acid transporter. These data support a mechanism wherein the nicotinamide glycolate ester serves as a lipophilic precursor that efficiently translocates into the intracellular neutrophil space to liberate the active acid form of the pharmacophore, which then acts at an intracellular site. Rapid inactivation by plasma esterases precluded use in vivo, but the mechanism elucidated provided insight for new nicotinamide pharmacophore classes with therapeutic potential.