CD94/NKG2C⁺ natural killer (NK) cells are increased in healthy individuals infected with human cytomegalovirus (HCMV), suggesting that HCMV infection may shape the NK cell receptor repertoire. To address this question, we analyzed the distribution of NK cell subsets in peripheral blood lymphocytes (PBLs) cocultured with HCMV-infected fibroblasts. A substantial increase of NK cells was detected by day 10 in samples from a group of HCMV⁺ donors, and CD94/NKG2C⁺ cells outnumbered the CD94/NKG2A⁺ subset. Fibroblast infection was required to induce the preferential expansion of CD94/NKG2C⁺ NK cells that was comparable with allogeneic or autologous fibroblasts, and different virus strains. A CD94-specific monoclonal antibody (mAb) abrogated the effect, supporting an involvement of the lectinlike receptor. Purified CD56⁺ populations stimulated with HCMV-infected cells did not proliferate, but the expansion of the CD94/NKG2C⁺ subset was detected in the presence of interleukin-15 (IL-15). Experiments with HCMV deletion mutants indicated that the response of CD94/NKG2C⁺ NK cells was independent of the UL16, UL18, and UL40 HCMV genes, but was impaired when cells were infected with a mutant lacking the US2-11 gene region. Taken together the data support that the interaction of CD94/NKG2C with HCMV-infected fibroblasts, concomitant to the inhibition of human leukocyte antigen (HLA) class I expression, promotes an outgrowth of CD94/NKG2C⁺ NK cells.