The Ptx1 (pituitary homeobox 1) homeobox transcription factor was isolated as a transcription factor of the pituitary POMC gene. In corticotrope cells that express POMC, cell-specific transcription is conferred in part by the synergistic action of Ptx1 with the basic helix-loop-helix factor NeuroD1. Since Ptx1 expression precedes pituitary development and differentiation, we investigated its expression and function in other pituitary lineages. Ptx1 is expressed in most pituitary-derived cell lines and as is the related Ptx2 (Rieger) gene. However, Ptx1 appears to be the only Ptx protein in corticotropes and the predominant one in gonadotrope cells. Most pituitary hormone-coding gene promoters are activated by Ptx1. Thus, Ptx1 appears to be a general regulator of pituitary-specific transcription. In addition, Ptx1 action is synergized by cell-restricted transcription factors to confer promoter-specific expression. Indeed, in the somatolactotrope lineage, synergism between Ptx1 and Pit1 is observed on the PRL promoter, and strong synergism between Ptx1 and SF-1 is observed in gonadotrope cells on the βLH promoter but not on the αGSU (glycoprotein hormone α-subunit gene) and βFSH promoters. Synergism between these two classes of factors is reminiscent of the interaction between the products of the Drosophila genes Ftz (fushi tarazu) and Ftz-F1. Antisense RNA experiments performed in αT3–1 cells that express the αGSU gene showed that expression of endogenous αGSU is highly dependent on Ptx1 whereas many other genes are not affected. Interestingly, the only other gene found to be highly dependent on Ptx1 for expression was the gene for the Lim3/Lhx3 transcription factor. Thus, these experiments place Ptx1 upstream of Lim3/Lhx3 in a cascade of regulators that appear to work in a combinatorial code to direct pituitary-, lineage-, and promoter-specific transcription.