We describe three novel genes, encoding members of the Toll-like receptor (Tlr) family (TLR7, TLR8, and TLR9). These Tlr family members, unlike others reported to date, were identified within a genomic database. TLR7 and TLR8 each have three exons, two of which have coding function, and lie in close proximity to one another at Xp22, alongside a pseudogene. The remaining gene (TLR9) resides at 3p21.3 (in linkage with the MyD88 gene), and is expressed in at least two splice forms, one of which is monoexonic and one of which is biexonic, the latter encoding a protein with 57 additional amino acids at the N-terminus. The novel Tlrs comprise a cluster as nearest phylogenetic neighbors. Combining all sequence data related to Toll-like receptors, we have drawn several inferences concerning the phylogeny of vertebrate and invertebrate Tlrs. According to our best estimates, mammalian TLRs 1 and 6 diverged from a common mammalian ancestral gene 95 million years ago. TLR4, which encodes the endotoxin sensor in present-day mammals, emerged as a distinct entity 180 million years ago. TLRs 3 and 5 diverged from a common ancestral gene approximately 150 million years ago, as did Tlr7 and Tlr8. Very likely, fewer Tlrs existed during early vertebrate evolution: at most three or four were transmitted with the primordial vertebrate line. Phylogenetic data that we have adduced in the course of this work also suggest the existence of a Drosophila equivalent of MyD88, and indicate that the plasma membrane protein SIGIRR is close functional relative of MyD88 in mammals. Finally, a single present-day representative of the Toll-like proteins in Drosophila has striking cytoplasmic domain homology to mammalian Tlrs within the cluster that embraces TLRs 1, 2, 4, and 6. This would suggest that an ancestral (pre-vertebrate) Tlr may have adopted a pro-inflammatory function 500 million years ago.