The POU-homeodomain transcription factor Pit-1 is required for the differentiation of the anterior pituitary cells and the expression of their hormone products. Pit-1beta, an alternate splicing isoform, has diametrically different outcomes when it is expressed in different cell types. Pit-1beta acts as a transcriptional repressor of prolactin (PRL) and growth hormone genes in pituitary cells, and as a transcriptional activator in non-pituitary cells. In order to explore these differences, we: (1) identified the transcriptional cofactors necessary for reconstitution of repression in non-pituitary cells; (2) tested the effect of the beta-domain on heterodimerization with Pit-1 and physical interaction with the co-activator CREB binding protein (CBP); and (3) determined the beta-domain sidechain chemistry requirements for repression. Co-expression of both Pit-1 isoforms reconstituted the repression of the PRL promoter in non-pituitary cells. The beta-domain allowed heterodimerization with Pit-1 but blocked physical interaction with CBP, and specific chemical properties of the beta-domain beyond hydrophobicity were dispensable. These data strongly suggest that Pit-1beta represses hormone gene expression by heterodimerizing with Pit-1 and interfering with the assembly of the Pit-1-CBP complex required for PRL promoter activity in pituitary cells.