The avian B cell differentiation antigen chB1 is a C-type lectin membrane protein most homologous to mammalian CD72. Here, we report a new chB1-related gene, chB1r, that is located 18 kb away the chB1 gene. The cytoplasmic domain of chB1r protein contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs: ITIM1 and 2), which are identical to those found in CD72, whereas chB1 lacks the second ITIM2. Although chB1 expression is restricted to the bursa and an immature B cell line, chB1r is highly expressed in the bursa, spleen and both immature and mature B cell lines, a pattern that parallels CD72 expression. SHP-1 and Grb2 interact with phosphorylated tyrosine residues within chB1r ITIM1 and ITIM2, respectively. By contrast, ITIM1 of chB1 does not interact with SHP-1. Functional characterization using chB1r/chB1 double-deficient DT40 B cells demonstrated that ITIM1 in chB1r transduces a negative signal for BCR-mediated nuclear factor of activated T cells (NF-AT) activation and that ITIM2 attenuates this negative signal. This study has established chB1r as the genuine avian homologue of mammalian CD72, and revealed an opposing role for the two ITIMs through binding with SHP-1 and Grb2 for regulation of BCR-mediated NF-AT activation.