Three different transcripts of the homeodomain gene termed pituitary homeobox (Ptx) 2 (Pitx2/Brx/Rieg/Solurshin/Arp) were cloned from different species encoding proteins belonging to the paired-like family of homeodomain proteins. Ptx2a (324 amino acids), Ptx2b (271 amino acids), and Ptx2c (318 amino acids) share the C terminus, including the homeodomain, and have different N termini. Here we report the comparative analysis of all three different Ptx2 splice variants for their transcriptional activity and their expression pattern in the adult rat brain. Ptx2 is able to trans-activate via different model promoters in different cell lines. A mild difference in trans-activating potential is observed among the splice variants, but the underlying mechanism is at present unknown. It is surprising that all Ptx2 transcripts displayed an identical expression pattern in the brain. This markedly restricted pattern is limited to the following brain areas: the anterior and intermediate lobes of the pituitary gland, the subthalamic nucleus, the posterior hypothalamic nucleus, the mammillary bodies, the red nucleus, and the deep gray layer of the superior colliculus. The data presented suggest that all variants of Ptx2 are involved in the development and regulation of distinct neuronal cell groups and the pituitary gland.