CD160/BY55 is a glucosyl-phosphatidylinositol (GPI)-anchored cell membrane receptor that is expressed primarily in natural killer (NK) cells. Its presence in CD8(+) T lymphocytes is considered to be a marker of cytotoxic activity, although there are few data in this regard. In the present work, we analysed the expression of CD160 in subpopulations of cytomegalovirus (CMV)-specific CD8(+) T cells. Subpopulations were defined by CD28 and CD57 expression and exhibited varying degrees of differentiation and cytotoxic potential, as evaluated by the expression of perforin, interferon (IFN)-gamma and interleukin (IL)-7Ralpha/CD127. We included subjects with different intensities of anti-viral immune response. Results showed that the terminally differentiated CD28(-) CD57(+) subset displaying the highest level of perforin expressed CD160 at a level similar to that of memory CD28(+) CD57(-)perforin(-) cells. A comparison of the expression of perforin in CD160(+) cells versus CD160(-) cells showed that expression was significantly higher in the absence of CD160. Interestingly, the CMV-specific CD8(+) T cell subset from a patient with ongoing CMV reactivation did not begin to express CD160 until day +92 of the follow-up period. Taken together, our data show that CD160 cannot be considered a cytotoxic marker in CMV-specific CD8(+) T cells.