We investigated the expression of bcl-2 oncoprotein in human cells by immunocytochemistry using an anti-bcl-2 antibody. Expression of bcl-2 was detected in and around mitotic nuclei and chromosomes from prophase to early telophase in all types of long-term cultured cell lines irrespective of the level of its expression in the cytoplasm. Western blot analysis showed a discrete but distinct protein band, which corresponded to the 26kDa α-form of bcl-2, in the lysate of cells showing only mitotic bcl-2. No other immunoreactions, including those corresponding to the β-form, were detected. RT-PCR combined with nested PCR showed bcl-2 mRNA in the same cell lines and in situ hybridization confirmed its presence in the cytoplasm of almost all interphase cells. The appearance of nuclear bcl-2 was not due to aberrant genetic regulation induced by an artificial in vitro environment. It was also not unique to neoplastic cells as nuclear and chromosomal bcl-2 was detected in freshly prepared specimens from B-cell lymphomas, myelodysplastic syndrome, and primary cultures of normal fibroblasts during mitosis. On the other hand, the level of cytoplasmic bcl-2 varied from one cell line to another; 8 carcinoma lines did not express cytoplasmic bcl-2, whereas many B cell lines expressed high levels of the protein. Bcl-2 appeared to accumulate in mitotic nuclei, because immunocytochemical reaction product was always stronger in mitotic nuclei than in the cytoplasm when the expression of bcl-2 was induced in peripheral blood lymphocytes by ConA stimulation. Our results indicate that bcl-2 protein is ubiquitously expressed in all cells around mitotic nuclei or chromosomes, whereas its distribution in the cytoplasm seems to reflect differences in genetic regulation based on cell lineage, differentiation stage or neoplastic state.