Bone marrow (BM) Ang II (angiotensin II) is a major participant in the regulation of hematopoiesis and immunity. The novel tissue substrate Ang-(1-12) [angiotensin-(1-12)] and its cleaving enzyme chymase are an essential source of Ang II production in cardiac tissue. We hypothesized this noncanonical chymase-mediated Ang II-producing mechanism exists in the BM tissue. Immunohistostaining and flow cytometry confirmed the presence of Ang-(1-12) immunoreaction in the BM of SD (Sprague Dawley) rats. Chymase-mediated Ang II-producing activity in BM was ≈1000-fold higher than ACE (angiotensin-converting enzyme)-mediated Ang II-producing activity (4531±137 and 4.2±0.3 fmol/min per mg, respectively; n=6; P<0.001) and 280-fold higher than chymase activity in the left ventricle of 16.3±1.7 fmol/min per mg (P<0.001). Adding a selective chymase inhibitor, TEI-F00806, eliminated almost all 125I-Ang II production. Flow cytometry demonstrated that delta median fluorescence intensity of chymase in cluster of differentiation 68 positive cells was significantly higher than that in cluster of differentiation 68 negative cells (1546±157 and 222±48 arbitrary units, respectively; P=0.0021). Cluster of differentiation 68 positive and side scatter low subsets, considered to be myeloid progenitors, express the highest chymase fluorescence intensity in rat BM. Chymase activity and cellular expression was similar in both male and female rats. In conclusion, myeloid lineage cells, especially myeloid progenitors, have an extraordinary Ang II-producing activity by chymase in the BM.