Tetracyclines (Tc's) have anti-inflammatory properties unrelated to their antibiotic activities. Their anti-inflammatory property, in part, results from the ability of members of this family of antibiotic to inhibit neutrophil functions. There are marked differences in the ability of different Tc's to suppress neutrophils which may relate to their ability to cross the plasma membrane. To gain insight into the mechanism of Tc inhibition of neutrophils and the reason for the differences in antineutrophil effect of Tc's, we studied the flux and sequestration of Tc's in blood cells. Using centrifugation and a dibutylphthalate scrubber system we found that doxycycline (Dc) was rapidly taken up by blood cells reaching intracellular concentrations several times that found in the medium. Dc also rapidly effluxed when antibiotic loaded cells were placed in drug free medium. While Ca2+, Mg2+ nor protein separately were effective inhibitors of Dc influx, when divalent cations and proteins were combined Dc uptake was markedly suppressed. Tc uptake by blood cells ranked Dc greater than chlortetracycline = tetracycline greater than oxytetracycline, a ranking similar to that reported for neutrophil inhibition by members of the Tc family, suggesting that intracellular accumulation of drug is an important facet of Tc suppression of neutrophil function.