Mycobacterium avium is an intracellular organism which multiplies predominantly within human macrophages. This organism has previously been shown to induce apoptosis in human macrophages. With a view to identifying M. avium components that induce cell death in infected host cells, sonicated extracts of M. avium as well as individual components isolated from the M. avium sonicate were tested in various assays with a human monocytic cell line (THP-1). THP-1 cells incubated with M. avium sonicate showed significantly reduced viability after a 2-day exposure compared to control cells incubated with media alone. This effect was dose dependent, with only 6.6% ± 5.2% and 48.8% ± 10.3% of the cells being viable by trypan blue exclusion at 600 and 300 μg/ml, respectively. Control cells, on the other hand, exhibited a viability of 98.8% ± 1.0%. In addition, an 80% ammonium sulfate fraction of the M. avium sonicate and the previously characterized 68-kDa protein were found to have similar effects on THP-1 cells. In both cases, the reduction in viability was due to apoptosis characterized by chromatin condensation, DNA fragmentation by agarose gel electrophoresis, or terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL) and release of nuclear matrix protein (NMP) into the culture medium. M. avium sonicate-induced apoptosis of THP-1 cells was completely inhibited by the commonly used antioxidants pyrrolidinedithiocarbamate (PDTC) and butylated hydroxyanisole (BHA), indicating that the generation of free oxygen radicals may be responsible for inducing cell death. M. avium sonicate was found to induce apoptosis of monocyte-derived macrophages (MDMs) as well. This effect was not reversed in the presence of PDTC and was not accompanied with DNA fragmentation when determined by agarose gel electrophoresis, as seen in the case of THP-1 cells. However, these MDMs were found to contain fragmented DNA by TUNEL. These findings suggest that the mechanism of cell death in MDMs may be different from that observed with THP-1 cells. Furthermore, these results provide new insight into the effect of M. avium components on host cell responses during M. avium infection.