Dendritic cells (DC) are efficient antigen presenting cells with the ability to activate naïve T cells. Murine DC represent a heterogeneous population that can be subdivided into distinct subsets, including the conventional DC (cDC) which are either CD4(-)CD8(-) (DN), CD4(+)CD8(-) (CD4+) or CD4(-)CD8(+) (CD8+) subsets and the plasmacytoid DC (pDC), which have different immune regulatory functions. In this study, we investigated the differential expression of genes encoding the inflammatory chemokines Mip-1alpha, Mip-1beta and Rantes, and the secretion of these chemokines, among splenic DC subsets. These chemokine genes were expressed at higher levels by the splenic CD4+ and DN cDC subsets compared with the CD8+ cDC, in both the resting and activated states in vivo. Both the pDC and cDC subsets displayed increases in chemokine secretion in response to a range of toll-like receptor (TLR) stimuli in vitro. Whilst the pDC were the highest producers of Mip-1alpha and Mip-1beta in response to some TLR stimuli, the DN and CD4+ cDC subsets were the superior producers of Rantes. Overall, of the cDC, the CD4+ cDC produced all chemokines most efficiently, both at a basal level, and in response to most TLR stimuli. Thus, we report a new functional difference between the murine splenic cDC subsets, with the CD4+ cDC demonstrating the most efficient production of the inflammatory chemokines Mip-1alpha, Mip-1beta and Rantes.