Cellular immunity is thought to be of major importance in resistance to infection with Mycobacterium paratuberculosis, the causative agent of Johne's disease of ruminants. The results of this study clarify the influence of bovine mononuclear phagocyte maturation on the ingestion and intracellular survival of M. paratuberculosis in vitro. Optimal phagocytosis of M. paratuberculosis by cultured bovine blood monocytes and monocyte-derived macrophages required the presence of 5 to 20% serum; few bacilli were phagocytized in the absence of serum. Monocyte-derived macrophages consistently demonstrated greater phagocytosis of M. paratuberculosis than did freshly adherent monocytes. Ingested M. paratuberculosis multiplied approximately 200 to 250% over a 7-day incubation period within bovine monocytes and monocyte-derived macrophages, as determined by microscopic counts of acid-fast-stained monocyte monolayers and by plate counts of viable organisms. These findings suggest that resident macrophages and recently emigrated blood monocytes within the intestinal mucosa may have considerable ability to ingest M. paratuberculosis, but they are unlikely to kill or markedly restrict the intracellular growth of the ingested bacilli. The ability of these mononuclear phagocytes to provide an intracellular niche for the growth of M. paratuberculosis and the immune response that eventually develops are likely to be important components in the development of the granulomatous lesions that are characteristic of Johne's disease.