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 CD205(Hi) and CD205(Lo) subpopulations which were phenotypically distinct and differed significantly (P < 0.01) in TLR gene expression. CD205(Hi) and CD205(Lo) mDCs were more efficient in macropinocytosis than monocytes and expressed no or little detectable non-specific esterase activity. CD205(Lo) 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 CD205(Lo) mDCs was associated with differential up-regulation of CD40, CD80, CD86 and TGF beta 1 gene expression when compared to CD205(Hi) 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. (C) 2014 Elsevier Ltd. All rights reserved.