Abstract The majority of folate uptake into tissues and tumors involves facilitated carriers, the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). PCFT is highly expressed in a wide range of human tumor cell lines and primary tumor specimens from a variety of lineages, with modest expression in most normal tissues. PCFT transports optimally at pHs characterizing the tumor microenvironment. We discovered novel 6-substituted pyrrolo[2,3-d]pyrimidine folate analogs with substantial selectivity for PCFT over RFC, resulting in potent antitumor efficacy in vitro and in vivo. However, in some cases PCFT levels varied substantially. Studies were performed to characterize the basis for these dramatic differences in PCFT gene expression between HepG2 (elevated) and HT1080 (negligible) tumor cells. Low levels of PCFT gene expression in HT1080 cells were not likely due to methylation of CpG islands in the PCFT promoter since 5-aza-2’-deoxycytidine treatment did not restore PCFT gene expression. A PCFT promoter reporter construct -2005/+96 in pGL3 (transcription starts at +1) was generated and showed high levels of activity in HeLa cells by luciferase reporter assays. For HepG2 and HT1080 cells, wide variations in PCFT transcript levels by real-time RT-PCR were paralleled by reporter gene activities with the -2005/+96 construct, suggesting that these were primarily due to PCFT transcriptional regulation. Progressive deletion analysis of the -2005/+96 construct and reporter assays in HepG2 and HT1080 cells localized the minimal promoter region to between -50 and +96. Additional deletion constructs (-35/+96, -20/+96, -15/+96 and -10/+96) were generated and studied. Whereas the -35/+96, -20/+96, -15/+96 promoter constructs retained ~50% activity for HepG2 cells and ~10-15% activity for HT1080 cells relative to the -50/+96 construct, in both cases the -10/+96 construct was essentially inactive. Two critical cis elements were identified in the PCFT minimal promoter by deletion analysis located between positions -50 and -35, and -15 and -10. Bioinformatics analysis localized putative cis elements for KLF-15 (-50 to -35) and NRF1 (-15 to -10) binding. The functional importance of these elements was confirmed by mutations of core consensus sequences in the -50/+96 construct and reporter gene assays. Binding of NRF1 and KLF15 to the endogenous PCFT promoter in HepG2 and HT1080 cells was confirmed by ChIP assays which paralleled patterns of differential PCFT gene expression. Collectively, our results suggest that NRF1 and KLF15 contribute to dramatic variations in PCFT gene expression between tumor cells. Better understanding the key determinants of PCFT transcriptional control may lead to strategies for modulating PCFT levels in combination with PCFT-targeted 6-pyrrolo[2,3-d]pyrimidine folate analogs for treating solid tumors. Citation Format: Zhanjun Hou, Carrie O'Connor, Steve Orr, Larry H. Matherly. Identification of transcriptional controls responsible for differential gene expression of the proton-coupled folate transporter in human solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5491. doi:10.1158/1538-7445.AM2017-5491