Pit-1 is a pituitary-specific transcription factor that plays a critical role in the normal development of the anterior pituitary gland. Previous analyses have shown that this protein exists in the rat pituitary gland and in rat pituitary-derived cell lines as two forms of relative molecular mass 33 and 31 kDa. This aspect of Pit-1 expression has been conserved throughout the evolution from rodents to humans. Here, we determine the origin of these structurally distinct forms of Pit-1 protein and find that these arise as a consequence of the alternative usage of translation initiation sites present in Pit-1 mRNA.