Acidosomes are organelles that in Paramecium are responsible for the acidification of phagosomes before phagosomes fuse with lysosomes. Using a combination of (a) the quick-freeze deep-etch (QF-DE) technique, (b) monoclonal antibodies (mAbs) that label specific membrane pools including those of the acidosomes, and (c) horseradish peroxidase (HRP)-uptake studies, we followed the development of acidosomes from the Golgi complex as well as the rapid transfer of HRP into the acidosomes. We also studied some of the characteristics of the involved membrane pools. Morphologically, acidosomes were first detected in the cytosol near the ER and Golgi stacks as clumps of tubules and vesicles, which apparently coalesced to form larger spherical or elongated preacidosomes. These clumped vesicles and preacidosomes had a QF-DE morphology resembling that of the mature acidosomes and were specifically labeled with mAbs that also labeled mature acidosomes. Within 10 s HRP cargo could be internalized by acidosomes while they were docked at the nascent vacuole membrane. This rapid uptake of HRP along with membrane occurs by vesicle fusion, a conclusion supported by QF-DE images. Thus the acidosome obtains its membrane from at least two sources, from the trans-Golgi network, and from the small HRP-containing vesicles. Cargo can also be acquired from two sources, the Golgi apparatus and the transport vesicles. Since it acquires non-particulate exogenous marker we conclude that the acidosome is linked to the endocytic pathway.