Human defensins are 29 to 30 amino acid (aa) antimicrobial peptides that are among the principal constituents of the neutrophil's azurophil granules. To determine the tissue specificity of posttranslational processing and subcellular targeting of defensins, the cDNA for a 94 aa human preprodefensin was transduced into murine cell lines (NIH 3T3 embryonic fibroblasts, AtT-20 pituitary adenoma, J774.1 and RAW 264.7 macrophages, and 32D and 32D cl3 granulocytes) using retroviral vectors. All transduced cell types expressed and to a variable extent constitutively secreted a 75 aa prodefensin formed by the removal of the amino terminal signal sequence. In AtT-20 cells, the 75 aa form accumulated intracellularly in granules and was releasable by secretagogues. Proteolytic processing to mature defensins was seen only in myeloid cells (J774.1, RAW 264.7, 32D, and 32D cl3). Newly formed mature defensin was rapidly degraded in J774.1 and RAW 264.7 macrophages, but accumulated stably in multivesicular bodies in 32D cells and in cytoplasmic granules of 32D cl3 cells. Our data suggest that the enzymatic and transport machinery required to process preprodefensin to mature defensin and to store it in cytoplasmic granules is a specialized feature of cells of granulocytic lineage.