An in vitro system was developed for studying host-parasite cellular interactions in visceral leishmaniasis with amastigotes isolated from infected spleens of hamsters and their peritoneal macrophages maintained by an improved method. The culture system supports the growth of Leishmania donovani amastigotes with different parasite/macrophage ratios for up to 2 wk, yielding results more consistent and reproducible than previously possible. Results indicated that the forms of the amastigotes (with or without adherent host membranes) and the state of the macrophages (with or without stimulation in vivo by thioglycollate or in vitro by aging) had no effect on the growth rate of the parasites, which, however, seems to vary with the macrophage subpopulations. An electron microscope study suggests that amastigotes are ingested through phagocytosis by the macrophages and become lodged in loose phagosomes. Additional evidence with quantitative data is presented to support the earlier findings that phagosome-lysosome fusion occurs after the interiorization of the parasites and that they not only survive but multiply in these vacuoles. During the postinfection periods, reorientation of amastigotes in vacuolar space results in the appearance of three types of parasitophorous vacuoles (parasites in loose vacuoles, in tight-fitting vacuoles or abutting in part against the inner lining of vacuoles). The last category may be the predominant type giving rise to the variations observed. Exogenously introduced dense marker accumulated in these parasitophorous vacuoles of the macrophages infected for several days indicating a continuous accessibility of amastigotes to the ambient mestruum via phagosome-lysosome vacuolar system of the host cells. This finding may have significant implications in parasite nutrition, host immunity, and chemotherapy of leishmaniasis.