The bcl-2 gene was originally cloned because of its involvement in B-cell lymphomas and encodes a 25-kD integral membrane protein that has been shown to inhibit programmed cell death (also termed apoptosis) in a wide variety of circumstances. The Epstein–Barr Virus (EBV) also has been implicated in B-cell malignancies and interestingly contains an open reading frame (BHRF-1) predicting a 19-kD protein with 22% homology to Bcl-2. To compare the functions of p26-Bcl-2 and p19-BHRF-1, we stably introduced expression plasmids encoding these proteins into a murine interleukin-3 (IL-3)-dependent hemopoietic cell line, 32D. Removal of IL-3 from cultures of control-transfected 32D cells resulted in internucleosomal DNA cleavage (a hallmark of programmed cell death) and loss of cell survival. In contrast, 32D cells containing high levels of p26-Bcl-2 or pl9-BHRF-2 proteins exhibited prolonged survival and markedly delayed DNA degradation under the same conditions of IL-3 deprivation. As a first attempt to determine the functional importance of amino acid sequences that are conserved between the Bcl-2 and BHRF-1 proteins, we used site-specific mutagenesis to replace two conserved cysteine residues with alanines (positions 158 and 219) in the human Bcl-2 protein. Comparisons of the wild-type and cysteine-minus human Bcl-2 proteins in S49 lymphoma cells revealed equivalent ability to block glucocorticoid-induced cell death and DNA fragmentation, indicating that these two conserved cysteines are not critical for Bcl-2 oncoprotein function. Investigations in 32D cells of an avian homolog of Bcl-2 cloned from the chicken also revealed conservation of function with the human Bcl-2 protein, despite the presence of a 48-amino-acid region of divergent sequence. Taken together, these data demonstrate that despite marked differences in their predicted amino-acid sequences, the human, chicken, and EBV versions of Bcl-2 have retained the structural characteristics necessary to interface with pathways involved in the regulation of programmed cell death in murine cells. The findings thus contribute to the mapping of functional domains in Bcl-2 proteins, and raise the possibility that the EBV-encoded pl9-BHRF-1 protein may be able to substitute for p26-Bcl-2 in the development of some types of cancer.