We have identified and characterised novel members of the mammalian polyhomeotic gene family, comprising a third human homologue, PHC3, and its mouse counterpart, Phc3. The two new genes have essentially the same genomic organisation, with 15 exons specifying proteins of 983 (PHC3) or 981 (Phc3) amino acids, with an overall sequence identity of 93%. Northern blot hybridisations have identified a single large transcript in a variety of adult mouse tissues and in situ hybridisation analyses indicate apparently ubiquitous expression during early human and mouse development. Exhaustive database screening suggests a maximum of three polyhomeotic homologues in humans and in mice and, for completeness, we have also derived the full-length coding sequence of the PHC2 gene (formerly called EDR2). The availability of full-length coding sequences for PHC2, PHC3 and Phc3 completes the profile of human and mouse polyhomeotic homologues and has enabled comprehensive comparative analyses of the human, mouse and Drosophila polyhomeotic proteins. The mammalian paralogues are located on three different chromosomes and pairwise comparisons of their protein products typically show about 34% amino acid sequence identity, except for some highly conserved domains for which we present consensus mammalian sequences. The data indicate that the novel PHC3 and Phc3 proteins are significantly more closely related to PHC2/Phc2 than either is to PHC1/Phc1 and reveal two hitherto unknown but highly conserved N-terminal domains that are shared by the PHC2/Phc2 and PHC3/Phc3 proteins but that are poorly conserved or absent in other polyhomeotic family members.