The biosynthesis of myeloperoxidase was studied in human myeloid leukemia HL-60 cells. Cells were pulse-labeled with [35S]methionine and cell extracts were treated with anti-myeloperoxidase serum. The immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a 1-h pulse, only two immunoreactive polypeptides of Mr 89,000 and 81,000 were labeled. During subsequent chase, the amounts of these polypeptides decreased with concomitant appearance of polypeptides of Mr 59,500 and 14,700, corresponding to the two chains of myeloperoxidase. Immunoprecipitation of all these polypeptides was prevented by adding excess myeloperoxidase. These results suggest that the Mr 89,000 and 81,000 polypeptides are precursors of myeloperoxidase. The precursors were present only in the soluble fraction of the cells, but the matured polypeptide chains were found in both the soluble and granule fraction after 17-h chase. Only one of the precursors, the Mr 89,000 peptide, was secreted into the culture medium.