Natural endogenous antisense RNAs have been reported in multiple loci, with evidence in some cases supporting a regulatory role for the antisense transcript. Here, we describe a novel gene, makorin RING zinc finger-2 (MKRN2), that overlaps and is antisense to the gene RAF1 in mammals. Phylogenetic analysis of the 3' untranslated region of RAF1 orthologues suggests that this relationship may have existed for up to 450 million years. We have also identified MKRN2 orthologues in two species of fish. This places the gene duplication event that created this locus from an ancestral MKRN1 gene early in vertebrate evolution, over 450 million years ago. Northern blot analyses show that human MKRN2 and RAF1 are co-expressed in tissues and cell lines, raising the possibility of mRNA duplex formation. The encoded makorin-2 protein is likely a ribonucleoprotein of unknown function, but its conservation suggests an important cellular role. The data presented here describe a conserved vertebrate MKRN2 gene that is closely associated with the RAF1 locus.