PKN is a newly discovered protein kinase that has been shown to mediate GTPase Rho dependent intracellular signalling. We show in this report that the mouse PKN gene is situated at the mouse EP₁prostanoid receptor gene locus and that the two genes are overlapping in a tail-to-tail manner. An “exon trap” strategy was used to identify the overlap phenomenon. By using RT-PCR and 3′ RACE we have identified two major PKN transcripts that are produced by alternative polyadenylation. The 3′ end of the short PKN transcript overlaps the 3′ untranslated region of the EP₁gene with ∼280 bp, while the long PKN transcript overlaps the whole EP₁gene. Remarkably, none of the three transcripts originating from this locus display the consensus AAUAAA polyadenylation signal. The last seven exons of the PKN gene, corresponding to the last third of the PKN cDNA, have been recognised in 7.2 kb of continuous genomic sequence that we have collected from the EP₁/PKN genetic locus. The 3′ part of the PKN gene is highly fragmented and its intron/exon organisation is reminiscent of that of theDrosophilaprotein kinase C gene. The possibility of a natural antisense regulation of these genes is discussed.