We have recently demonstrated that simian immunodeficiency virus (SIV) Nef binds to the zeta chain of the T-cell receptor (TCR), leading to its down-modulation from T-cell surfaces (I. Bell, C. Ashman, J. Maughan, E. Hooker, F. Cook, and T. A. Reinhart, J. Gen. Virol. 79:2717-2727, 1998). Using a panel of human as well as rhesus macaque TCR zeta cytoplasmic domain mutants, we have identified in this report two linear peptides in the cytoplasmic domain of TCR zeta which independently interact with SIV Nef. Each SIV Nef interaction domain was sufficient in the absence of the other for interaction with SIV Nef in a yeast two-hybrid assay. In parallel, we demonstrated that Nef down-modulation of CD8-TCR zeta fusion proteins containing full-length or truncated portions of the TCR zeta cytoplasmic domain occurs in transiently transfected 293T cells. Furthermore, using proteins expressed in Escherichia coli, a glutathione S-transferase-Nef fusion protein coprecipitated histidine-tagged portions of the TCR zeta cytoplasmic domain which contained SNID-1 or SNID-2. The peptides targeted by SIV Nef, YNELNL and YSEIGMKGERRR, are portions of the first and second of three immunoreceptor tyrosine-based activation motifs which are important in signal transduction, thymocyte development, and TCR biogenesis. These results demonstrate that SIV Nef binds to two distinct domains on TCR zeta in the absence of other T-cell-specific factors, and that interaction with either domain is sufficient to cause down-modulation of TCR zeta.