[3H]LTB4 binds concentration-dependently to intact human PMNs. The binding is saturable, reaches equilibrium in 10 min at 4 degrees C, and is readily reversible. Mathematical modeling analysis reveals biphasic binding of [3H]LTB4, indicating two discrete populations of binding sites. The high-affinity binding sites have a dissociation constant of 0.46 X 10(-9) M and a Bmax of 1.96 X 10(4) sites per neutrophil; the low-affinity binding sites have a dissociation constant of 541 X 10(-9) M and a Bmax of 45.16 X 10(4) sites per neutrophil. Competitive binding experiments with structural analogues of LTB4 demonstrate that the interaction between LTB4 and the binding site is stereospecific and correlates with the relative biological activity of the analogues. At 25 degrees C, [3H]LTB4 is rapidly dissociated from the binding site and metabolized to 20-OH- and 29-COOH-LTB4. Purification of neutrophils in the presence of 5-lipoxygenase inhibitors significantly increases specific [3H]LTB4 binding, suggesting that LTB4 is biosynthesized during the purification procedure. These data suggest that stereospecific binding and metabolism of LTB4 in neutrophils are tightly coupled processes.