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The endometrial expression of the gene encoding porcine uteroferrin (UF), during pregnancy is presumed to be mediated by cis-regulatory regions distinct from those that confer its limited expression to other mammalian tissues and cell types. In the present study, chimeric DNA constructs of native and progressive 5’ deleted promoter regions fused to the promoterless chloramphenicol acetyl-transferase reporter gene were transiently transfected in the human endometrial carcinoma cell line ECC-1 to examine their ability to direct UF promoter activity. The region between -1935 and -831 bp contained negatively acting elements which drastically reduced basal promoter activity. In contrast, the region between -831 and -484 bp contributed significantly to high level basal activity. Gel retardation and footprinting assays identified factor-binding sites between -1601 and -484 bp for human endometrial nuclear proteins. One binding site corresponds to a heptamer motif (TGCTAGA) present twice within the -1601 to -831 bp region and previously shown to bind an 80 kDa porcine endometrial protein. This heptamer bound an 80 kDa nuclear protein from human ECC-1 and human Ishikawa endometrial cells and a 92 kDa protein from human placental JEG-3 cells. The other binding region within -831 to -484 bp contained GC-rich sequences, which bind human Sp1. The protected GC-rich sequence (GC-Box 1) between -768 arid -749 bp also binds a 24 kDa M(r) protein. Nuclear proteins of molecular weight 40-60 kDa and distinct from Sp1, Sp2 and Sp3 bound a second GC-rich sequence (GC-Box 3) between -628 and -616 bp. These studies demonstrate that multiple elements within the UF gene promoter bind nuclear proteins which are similarly expressed in other endometrial cells and suggest that common transactivating factors may functionally mediate expression of endometrial-associated genes.