We previously reported that the number of acid-fast bacilli within Mycobacterium paratuberculosis-infected bovine monocytes increased steadily during an 8-day incubation period in vitro, despite a decrease in the number of viable bacilli as estimated by a radiometric method. In this study, we used differential live/dead staining of bacilli from infected monocytes to show that the percentage of viable bacilli decreased during an 8-day incubation period. We observed poor phagosome-lysosome fusion in monocytes that had ingested viable M. paratuberculosis (30% phagosome-lysosome fusion), while monocytes that ingested heat killed M. paratuberculosis exhibited 94% phagosome-lysosome fusion at 24h after infection. Treatment with the selective Ca(2+)/CaM and PI3 kinase inhibitors (i.e. KN62 and Wortmannin) in combination increased the survival of M. paratuberculosis in bovine monocytes without significantly altering phagosome-lysosome fusion. Scanning electron microscopy suggested that M. paratuberculosis-infected monocytes were less differentiated (smaller and less spreading) than uninfected monocytes at 4 and 8 days of infection. Overall, these data suggest that both multiplication and killing of intracellular M. paratuberculosis occur concomitantly in bovine monocytes. Monocytes in turn may be adversely affected by the bacilli, their products, or factors released from infected monocytes.