To further understand the pathogenesis of Mycobacterium avium ss paratuberculosis infection in the bovine, we investigated the phagocytosis by, and intracellular survival of, Mycobacterium avium ss paratuberculosis in bovine monocytes from non-infected cows and Mycobacterium avium ss paratuberculosis-infected cows. The different abilities of these cells to ingest and inhibit intracellular growth of M. avium ss paratuberculosis were determined either by radiometric (BACTEC) counting of bacilli in monolayer lysates, or by microscopic counting of bacilli in monocytes at 0, 3, 6, 9, 12, and 15 days post-infection in vitro. We observed an early growth phase, during the first 6 days after in vitro infection, followed by mycobacteriostasis or bacterial killing. This is the first report of decreasing numbers of viable intracellular M. avium ss paratuberculosis in bovine monocytes after 6 days of in vitro infection. In contrast, the numbers of acid-fast bacilli, as detected by microscopic evaluation, continued to increase, suggesting loss of viable bacilli concurrent with bacillary replication. This early growth phase suggests that M. avium ss paratuberculosis may initially resist antimycobacterial mechanisms of mononuclear phagocytes, or that these antimycobacterial mechanisms are not activated. The later period of mycobacteriostasis, or killing of bacilli, could be related to release of endogenous cytokines that lead to activation of bactericidal mechanisms (ROI, RNI), or nutrient deprivation of intracellular bacilli. Monocytes from infected cows with a strong IFN-gamma response phagocytosed slightly more bacilli than did monocytes of normal cows. There was a trend toward mycobacteriostasis or bacterial killing occurring somewhat earlier in monocytes from M. avium ss paratuberculosis infected cows than in monocytes from normal cows.