The phagosomes containing viable pathogenic mycobacteria, such as Mycobacterium (M.) tuberculosis and Mycobacterium avium ssp. avium (M. avium), are known to be limited in their ability to both acidify and fuse with late (but not early) endocytic organelles. Here, we analysed the pH and fusogenicity of phagosomes containing M. avium ssp. paratuberculosis (M. ptb), the causative agent of paratuberculosis in ruminants. Using the murine J774 macrophage cell line, we compared viable and heat-killed M. ptb and, in addition, viable or dead M. avium, as well as two non-pathogenic mycobacteria, Mycobacterium smegmatis and Mycobacterium gordonae. Electron microscopic analysis revealed that M. ptb persisted intracellularly in phagosomes for up to 15 days. The phagosomes containing live M. ptb and M. avium were significantly reduced in their ability to acquire some markers for the endocytic pathway, such as internalized calcein, BSA-gold or the membrane protein Lamp 2. However, they were almost completely accessible to 70 kDa fluorescein isothiocyanate (FITC)-dextran and Lamp 1. Overall, the phagosomes containing dead pathogenic mycobacteria behaved similarly to the ones containing live non-pathogenic mycobacteria in all experiments. Using FITC-dextran in a novel fluorescence-activated cell sorting (FACS)-based method, we could also show that the bulk of endocytic compartments, including phagosomes, were only very mildly acidified to approximately pH 6.3 over at least 72 h in J774 cells infected with live M. ptb and M. avium. In contrast, J774 cells treated with heat-killed M. ptb or BSA-coated latex beads showed substantial acidification of the phagosome/endocytic compartments to a pH value of approximately 5.2. After infection with M. smegmatis and M. gordonae, acidification was initially (1-5 h after infection) inhibited, but increased after longer infection to levels similar to those with dead mycobacteria.