Activation of human plasmacytoid dendritic cells (pDCs) with ligands for Toll-like receptors (TLRs) 7 and 9 induces the secretion of type I interferons and other inflammatory cytokines as well as pDC differentiation. Transcripts for 2 members of the CD300 gene family, CD300a and CD300c, were identified on pDCs during gene expression studies to identify new immunoregulatory molecules on pDCs. We therefore investigated the expression of CD300a and CD300c and their potential regulation of pDC function. CD300a/c RNA and surface expression were downregulated after stimulation of pDCs with TLR7 and TLR9 ligands. Exogenous interferon (IFN)-alpha down-regulated CD300a/c expression, whereas neutralizing IFN-alpha abolished TLR ligand-induced CD300a/c down-regulation. This implicates IFN-alpha in regulating CD300a/c expression in pDCs. In addition, IFN-alpha favored tumor necrosis factor (TNF)-alpha secretion by CpG-induced pDCs. CD300a/c triggering by cross-linking antibody reduced TNF-alpha and increased IFN-alpha secretion by pDCs. Furthermore, CD300a/c triggering, in the presence of neutralizing IFN-alpha, further reduced TNF-alpha secretion. These data indicate that CD300a and CD300c play an important role in the cross-regulation of TNF-alpha and IFN-alpha secretion from pDCs.