Mycobacterium avium is an intracellular pathogen able to invade and survive within macrophages and mucosal epithelial cells. The study of the interaction between M. avium and host cells is important to establish the mechanisms of pathogenesis of the infection. One of the limitations of microscopic study of intracellular M. avium is the difficulty of identifying isolated bacilli within cells. As a general strategy to visualize and analyse the influence of M. avium on intracellular trafficking, we cloned the gene encoding the green fluorescent protein (GFP) in M. avium. A vector was constructed by cloning the cDNA for the GFP of the bioluminescent jellyfish Aequorea victoria into an Escherichia coli/Mycobacteria shuttle vector (pMV261). The recombinant plasmid (pWES-4) was transformed into both E. coli and M. avium strain 104 (serovar 1). The transformants were screened for strong expression of the GFP. Transformed M. avium were clearly visible inside human macrophages and epithelial cells using fluorescence microscopy. These transformed M. avium should provide a useful tool for further study of intracellular behaviour.