The observation that myeloperoxidase precursor and larger intermediate (Mr 91,000 and 81,000, respectively) were extracted in the presence of detergent from isolated granule fractions of human promyelocytic leukemia HL-60 cells under mildly acidic conditions was investigated. In contrast, under conditions of neutral pH, only the Mr 74,000 intermediate and mature species were extracted. Extraction of the Mr 91,000 and 81,000 forms was also enhanced in the presence of EDTA. Kinetic studies of the processing of the different myeloperoxidase species confirmed the intermediate nature of the Mr 81,000 and 74,000 forms. Support for a role of an acidic intracellular compartment was obtained through evidence that the acid-extractable precursor and intermediates accumulated in HL-60 cells which had been treated with 1 microM monensin. Under these conditions, the production of mature heavy (Mr 63,000) and light (Mr 13,500) subunits of myeloperoxidase was consistently inhibited by greater than 40% over a 16-h period. The effects of monensin on processing of myeloperoxidase were completely reversed if monensin was removed during this 16-h period. These data support the idea that an acidic compartment may be involved in the transport of myeloperoxidase precursors to azurophil granules and/or their processing to a smaller intermediate form (Mr 74,000) of the enzyme.