Platelet dense granules (DGs) are storage organelles for calcium ions, small organic molecules such as adenosine 5′-diphosphate and serotonin, and larger polyphosphates that are secreted upon platelet stimulation to enhance platelet activation, adhesion, and stabilization at sites of vascular damage. DGs are thought to fully mature within megakaryocytes (MKs) prior to platelet formation. Here we challenge this notion by exploiting vital fluorescent dyes to distinguish mildly acidic DGs from highly acidic compartments by microscopy in platelets and MKs. In isolated primary mouse platelets, compartments labeled by mepacrine, a fluorescent weak base that accumulates in DGs, are readily distinguishable from highly acidic compartments, likely lysosomes, that are labeled by the acidic pH indicator LysoTracker and from endolysosomes and α granules labeled by internalized and partially digested self-quenching BODIPY–labeled bovine serum albumin (DQ BSA). By contrast, in murine fetal liver and human CD34+ cell-derived MKs and the megakaryocytoid cell lines, MEG-01 and differentiated G1ME2, labeling by mepacrine overlapped nearly completely with labeling by LysoTracker and partially with labeling by DQ BSA. Mepacrine labeling in G1ME2-derived MKs was fully sensitive to proton ATPase inhibitors but was only partially sensitive in platelets. These data indicate that mepacrine in MKs accumulates as a weak base in endolysosomes but is likely pumped into or retained in separate DGs in platelets. Fluorescent puncta that labeled uniquely for mepacrine were first evident in G1ME2-derived proplatelets, suggesting that DGs undergo a maturation step that initiates in the final stages of MK differentiation. Display omitted •Compartments labeled by the vital dense granule dye mepacrine are distinct from acidic structures in platelets but not in megakaryocytes.•Distinct hypoacidic mepacrine-labeled structures first appear during megakaryocyte differentiation to proplatelets.