Studying genetic diversity of immunologically relevant molecules can improve our knowledge on their functional spectrum in normal immune responses and may also uncover a possible role of different variants in diseases. We characterized the c.503T>C polymorphism in the human KLRB1 gene (Killer cell lectin-like receptor, subfamily B, member 1) coding for the cell surface receptor CD161. CD161 is expressed by subsets of CD4+ and CD8+ T cells and the great majority of CD56+ natural killer (NK) cells, acting as inhibitory receptor in the latter population. Genotyping a cohort of 118 healthy individuals revealed 40% TT homozygotes, 46% TC heterozygotes, and 14% carriers of CC. There was no difference in the frequency of CD161 expressing CD4+ and CD8+ T cells between the different genotypes. However, the frequency of CD161+ NK cells was significantly decreased in CC carriers as compared to TT homozygotes. c.503T>C causes an amino acid exchange (p.Ile168Thr) in an extracellular loop of the CD161 receptor, which is regarded to be involved in binding of its ligand Lectin-like transcript 1 (LLT1). Binding studies using soluble LLT1-Fc on 293 transfectants over-expressing CD161 receptors from TT or CC carriers suggested diminished binding to the CC variant. Furthermore, triggering of CD161 either by LLT1 or anti-CD161 antibodies inhibited NK cell activation less effectively in cells from CC individuals than cells from TT carriers. These data suggest that the c.503T>C polymorphism is associated with structural alterations of the CD161 receptor. The regulation of NK cell homeostasis and activation apparently differs between carriers of the CC and TT variant of CD161.