Myeloid-related protein 8 (MRP8) and MRP14 are S100 family calcium binding proteins that form a heterodimer known as MRP8/14 that is present in the cytosol of neutrophils and monocytes. MRP8/14 becomes associated with endothelium at sites of monocyte and neutrophil adhesion and transmigration and induces a thrombogenic and inflammatory response by increasing the endothelial transcription of proinflamatory chemokines and adhesion molecules. MPR8/14 is the most abundant protein in neutrophil cytosol making up 30 to 60% of all cytosolic protein. However, the distribution of MPR8/MRP14 among neutrophil granules and plasma membranes in unclear and was investigated to better understand the role or MPR8/14 in acute inflammation. Three monoclonal antibodies specific for MRP8 and MPR14 were characterized, AHN-17, ANH-17.1, and 15H9, and were used to investigate the subcellar distribution of MPR8/14. The 10 and 14 kDa proteins recognized by the three antibodies were isolated by affinity chromatography with 15H9 and were separated by reverse phase high pressure liquid chromatography. N-terminal amino acid sequencing of the 10 kDa protein yielded a sequence of 29 amino acids that was identical to MRP8. The N-terminus of the 14 kDa protein was blocked, however, the amino acid sequence of two tryptic peptides were found to be identical to MRP14 at 25 of 27 amino acids. Nitrogen cavitation and density gradient separation was used to isolate neutrophil cytosol, plasma membranes, primary granules, and secondary granules. The secondary granule fraction also contains a distinct granule population termed tertiary or secretory granules. The proteins recognized by AHN-17 were isolated from each of these fractions by affinity chromatography and analyzed by SDS-PAGE under reducing conditions. MRP8 and MRP14 were located in the cytosol, plasma membrane, and secondary granule, and to a lesser degree primary granule fraction. To further determine the location of MPR-8/14 in the cellular granules, primary and secondary granules were washed and disrupted by freeze-thawing and sonication and the membranes were separated from the granule contents by centrifugation and were analyzed by immunoblotting with 15H9. Both MRP8 and MRP14 were detected in the soluble portion of the secondary granules. MPR14 was also detected in the soluble portion of primary granules and MRP8 in the plasma membrane fraction. The largest quantities of the MRP8 and MRP14 were present in cytosol and in the soluble portion of the secondary granules. MRP8 and MRP14 had a loose calcium-dependent adherence to neutrophil primary granules and plasma membranes and they were removed by washing with EGTA in a high ionic strength buffer. In conclusion MRP8/14 is located in neutrophil cytosol and in the contents of secondary granules and is loosely associated with plasma membranes and primary granules. MRP8/14 released with secondary granules by activated neutrophils likely binds to endothelium and plays an important role in acute inflammation.