Myeloperoxidase (MPO), a characteristic enzyme of human polymorphonuclear neutrophils (PMN), is localized in specialized lysosomal or azurophilic granules, and can be resolved into three distinct forms (I, II, III) by ion-exchange chromatography. Granules were isolated from single donor PMN and fractionated with centrifugation into two different azurophilic subpopulations (high and low density) by banding in a continuous sucrose density gradient. Ion-exchange chromatography of granule extracts indicated that the lower density granules contained mainly MPO forms II and III while the higher density granules appeared to contain all three forms, but in much reduced amounts. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed that the mobilities of the heavy subunits of MPO appeared to be inversely related to the density of the granule population from which they were extracted. These observations suggest that the different forms of MPO may have distinct functional roles and/or are a possible reflection of maturational differences among the granule subpopulations.