The cDNA for eosinophil granule major basic protein (MBP) encodes a prepromolecule with a total length of 222 amino acids (preproMBP). PreproMBP includes a secretory leader of 15 amino acids, an acidic propiece of 90 amino acids, and a basic MBP portion of 117 amino acids. The function of the propiece, which has a predicted pI of 3.9, is unknown, but it gives proMBP an overall acidic charge. Because proMBP is not found in mature eosinophils, we analyzed eosinophil differentiation in interleukin-5 (IL-5)-stimulated umbilical cord stem cells cultured for 24 days. By immunofluorescence, proMBP appeared by day 6 and peaked on day 18, whereas MBP was prominent at days 12 to 24. By day 6, Western blots detected heterogeneous glycosylated 33-kD proMBP; its peak expression occurred on day 12. Western blots showed sequential processing of 33-kD proMBP to an 18-kD intermediate form and finally to 14-kD MBP. By dual label immunoelectron microscopy, proMBP was localized primarily to large uncondensed eosinophil granules, whereas MBP was localized to granules containing a condensed central area. Thus, proMBP is likely expressed and processed as the granule condenses in a multistep process to 14-kD MBP in differentiating eosinophils.