The homing properties of subsets of lymphocytes and dendritic cells (DC) are regulated in part by the profile of chemokine receptors expressed. To determine how CCR6 influences cell trafficking, a mutant allele of the mouse CCR6 gene was produced that includes an enhanced green fluorescent protein (EGFP) reporter under the control of the CCR6 promoter. In mice heterozygous for the EGFP/CCR6 knock-in, CCR6 expression was detected on all mature B cells, subpopulations of splenic CD4(+) and CD8(+) T cells, and on some CD11c(+) DC. Most CD11b(+) myeloid DC expressed CCR6, but CD8alpha(+) lymphoid DC were negative for CCR6. Among myeloid DC, the CD4(+) subset was uniformly positive for CCR6 expression and the CD4(-) subset was mostly CCR6 positive. Epidermal Langerhans cells (LC) also expressed CCR6, but at lower levels than splenic myeloid DC. Culture of bone marrow precursors from the knock-in mice with GM-CSF for 4 to 6 days led to the appearance of a subset of CD11c(+) DC expressing CCR6. The differences in CCR6 expression among the major DC subsets indicate that CCR6 and its chemokine ligand MIP-3alpha participate in determining the positioning of DC subsets in epithelial and lymphoid tissues.