Prophet of Pit-1 (Prop-1) is a paired class homeodomain transcription factor that is specifically expressed in the pituitary gland. Mutations in the Prop-1 gene cause compound pituitary diseases in mouse models and human patients. We have cloned and analyzed the porcine ortholog of Prop-1. Analysis of cDNAs revealed that the porcine Prop-1 sequence is similar to the mouse and human proteins within the homeodomain and carboxyl terminus, but the amino terminus is poorly conserved. The Prop-1 gene consists of three exons and two introns and spans 3.8 kilobases of genomic DNA. In addition, we mapped Prop-1 to the q arm of pig chromosome two. During development, Prop-1 is expressed at the time of pituitary organogenesis. In the adult, expression was observed at low levels only in the pituitary gland. The porcine Prop-1 protein displays similar biochemical, DNA binding, and transcriptional activities to human PROP-1. We conclude that, although the structural divergence between the porcine and human PROP-1 molecules may indicate some distinct functions, the porcine Prop-1 gene encodes a pituitary transcription factor with similar overall activities to the human ortholog.