Introduction: The gut-enriched Krüppel-like factor (KLF-4) and the liganded thyroid hormone receptor (TRα1) have been shown to activate intestine-specific genes and each is thought to play a critical role in gut development and differentiation. We investigated an inter-relationship between these two presumably independent pathways using the differentiation marker gene, intestinal alkaline phosphatase (IAP). Methods: Transient transfections were performed in Cos-7 cells using luciferase reporter plasmids containing a 2.5 kb segment of the human IAP 5' regulatory region, as well as multiple deletions. Cells were transfected with TRα1 and/or KLF-4 expression vectors and treated -/+ 100 nM thyroid hormone (T3). Results: IAP transcription was activated independently by KLF-4 (8.1 fold) and ligand-bound TRα1 (4.7 fold). Cells co-transfected with KLF-4 and TRα1 in the presence of T3 showed a 41.7 fold increase (p<0.05). In the absence of T3 the degree of activation was similar to KLF-4 alone (10.3 fold). Additionally, the synergistic effect was lost with a specific internal deletion (-224/-114) in the IAP regulatory region. Conclusions: The thyroid hormone receptor and KLF-4 synergistically activate the enterocyte differentiation marker intestinal alkaline phosphatase, suggesting a previously unrecognized inter-relationship between these two transcription factor pathways.