Technical Abstract: Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a facultative intracellular pathogen that resides within host macrophages during infection of ruminant animals. The objective of this study was to define the kinetics of M. paratuberculosis growth and survival within J774.16 macrophages. Serial plating of M. paratuberculosis infected macrophage lysates on Herold's egg yolk medium showed that mycobacterial replication takes place between 0 and 24 hours post-infection. This initial growth phase was followed by a slow decline in viability over the next six days. Electron microscopy demonstrated that initial intracellular bacterial growth was the consequence of simultaneous intracellular killing and replication of bacteria within macrophages as degraded mycobacteria were observed even at early times postinfection. Immunogold labeling of infected macrophages with antibody against M. paratuberculosis showed degraded intracellular mycobacteria that were unrecognizable by morphology alone. When macrophages were heavily infected with M. paratuberculosis, no degraded forms were observed and macrophages were killed, presumably by apoptosis. We present a general description of M. paratuberculosis survival within cultured macrophages using transmission electron microscopy and other techniques.