Mycobacterium avium is an opportunistic pathogen associated with pulmonary disease in non-AIDS patients and disseminated infection in patients with AIDS. The chief route of infection is by colonization and invasion of the mucosa of the gastrointestinal tract, but infection through the respiratory route also occurs. After crossing the mucosa, M. avium infects and replicates within tissue macrophages. To identify M. avium genes required for survival in vivo, a library of signature-tagged transposon mutants was constructed and screened for clones attenuated in mice. Thirty-two clones were found to be attenuated for their virulence, from which eleven were sequenced and tested further. All the mutants studied grew similarly in vitro to the wild-type MAC104. Ten mutants were tested individually in mice, confirming the attenuated phenotype. MAV_2450, a polyketide synthase homologue to Mycobacterium tuberculosis pks12, was identified. STM5 and STM10 genes (encoding two hypothetical proteins MAV_4292 and MAV_4012) were associated with susceptibility to oxidative products. Mutants MAV_2450, MAV_4292, MAV_0385 and MAV_4264 live in macrophage vacuoles with acidic pH (below 6.9). Mutants MAV_4292, MAV_0385 and MAV_4264 were susceptible to nitric oxide in vitro. The study of individual mutants can potentially lead to new knowledge about M. avium pathogenic mechanisms.