Immature myeloid (m)DCs circulating in the blood of cattle have been defined as lineage negative (Lin−)MHCII+CD11c+CD205+ cells. Lin−MHCII+CD11c+CD205+ mDCs (0.2% blood mononuclear cells) isolated from bovine blood were heterogeneous in cell size and CD205 expression. Using highspeed cell sorting, Lin−MHCII+CD11c+CD205+ DCs were sorted into CD205Hi and CD205Lo subpopulations which were phenotypically distinct and differed significantly (P < 0.01) in TLR gene expression. CD205Hi and CD205Lo mDCs were more efficient in macropinocytosis than monocytes and expressed no or little detectable non-specific esterase activity. CD205Lo mDCs efficiently activated purified allogeneic T cells and the addition of TLR agonists did not significantly alter this antigen presentation capacity. T cell activation by CD205Lo mDCs was associated with differential up-regulation of CD40, CD80, CD86 and TGFβ1 gene expression when compared to CD205Hi mDCs. In conclusion, two phenotypically and functionally distinct CD11c+CD205+ mDCs were isolated from blood that had an equal capacity to acquire antigen but markedly different capacities to activate T cells.