Azurophil granules of myeloid cells form in promyelocytes. They store cytotoxic and digestive agents which when released are involved in the defense against infection. In order to characterize the intragranular distribution of these agents, ultrastructural methods using immunogold were used on promyelocytes. Azurophil granules were divided into nucleated, large spherical (large azurophil) and small electron-dense (small azurophil) granules. Myeloperoxidase showed a peripheral distribution of large azurophils and a uniform distribution of small and nucleated azurophils, consistent with previous findings. Likewise, the major neutral proteases of azurophils, cathepsin G, granulocyte elastase, and proteinase 3, displayed a similar distribution, with a peripheral localization in large azurophils and a uniform distribution in small and nucleated azurophils, except for proteinase 3, which was associated with the crystalloid structure in nucleated azurophils. In contrast, the bactericidal/permeability increasing protein, which is bacteristatic and bactericidal for Gram-negative bacteria, was localized to the membrane area in all types of azurophil granules, consistent with a suggested association of this protein with the granule membrane. The observed differences in intragranular distribution of the proteins investigated may reflect variations in binding to matrix structures and granule membranes.