The cell-specific actions for prolactin (PRL) are dependent on the coordinated transcriptional regulation of the prolactin receptor (PRLR) gene across tissues. Several lines of evidence indicate that the use of multiple promoters underlies the tissue specific regulation for PRLR expression in states such as pregnancy and lactation. However, the role and regulation of PRL/PRLR action in the kidneys is less clear. We previously identified that one of several first exons of the porcine PRLR is expressed exclusively in the kidneys, small intestine, and liver. We found the 5’ non-coding region of this first exon (E1.3) is conserved across mammalian PRLR, including ungulates and humans, and its expression increased throughout gestation. We hypothesized E1.3 expression in the kidney was regulated by tissue-specific factors acting on the E1.3 promoter (promE1.3), and its expression increased in support of osmoregulation. Our objectives were to 1) resolve the factor(s) regulating E1.3 expression in the kidneys of pigs and 2) develop an ex vivo model to study E1.3 regulation in the kidneys. We found promE1.3 activation was tissue and cell line specific, whereby the proximal promoter was only active in kidney (LLC-PK1) and colorectal (Caco-2) cells. We also found an element downstream of the transcription start site (TSS) that acts to repress promoter activation in all cell lines tested. In silico transcription factor binding analysis of the proximal promoter revealed multiple consensus sequences for tissue-specific transcription factors, including hepatocyte nuclear factor 1 (HNF1). Mutation of one of the binding sites for HNF1 within the E1.3 promoter resulted in a significant (p<0.05) downregulation in promoter activity in Caco-2 cells, and a reduction in promoter activity in LLC-PK1 cells. Using our kidney explant model, we found E1.3 was more highly expressed than the ubiquitous first exon of the PRLR (exon 1A, E1A) ex vivo. Interestingly E1.3 expression significantly (p<0.05) declined with time in culture, and E1A became the predominant transcript expressed after 48 hours (h). There was no significant difference in PRLR long form or short form expression over time in culture. However, mRNA expression for HNF1 and HNF4 variants declined (p<0.05) over 48h in culture. Together, these data demonstrate that elements within the proximal promoter region of promE1.3 are vital for E1.3 expression, and tissue-specific elements may be required for its sustained expression. It remains to be determined if HNF transcription factors are directly or indirectly responsible for the regulation of PRLR expression in the kidneys of pigs.