Placental growth factor (PGF, previously known as PlGF) is prominently expressed by trophoblasts in human placenta, whereas most nontrophoblast cells express low levels of PGF mRNA under normal physiological conditions. We have shown that hypoxia decreases PGF expression in the trophoblast, but little is known about transcriptional regulation of PGF gene expression. We sought to determine promoter regions of the human PGF gene that contribute to its restricted high constitutive expression in the trophoblast. Overlapping putative promoter regions of human PGF gene encompassing 2-1.5 kb were cloned into reporter vectors and co-transfected into trophoblast and nontrophoblast cell lines. Promoter activity generated by a 2-1.5-kb clone was significantly higher in trophoblasts than in nontrophoblasts. Selective deletion mutants showed that a clone encompassing the PGF (2-828/++34) region generated promoter activity similar to the 2-1.5-kb region in the trophoblast. However, deletion of another 131 bp from this subclone (2-698/++34) resulted in significantly less promoter activity in the trophoblast. The (2-828/2-698) region significantly enhanced activity of a minimal promoter construct in trophoblast but not in nontrophoblast cells, suggesting that this region contributes to regulating PGF transcription in the trophoblast. Site-directed mutagenesis of a glial cell missing 1 (GCM1) motif in the 131-bp region significantly decreased enhancer activity in the trophoblast. Furthermore, overexpression of GCM1 significantly increased PGF 2-1.5-kb promoter activity and PGF mRNA expression in trophoblast and nontrophoblast cells. Forced overexpression of GCM1 restored PGF expression in the hypoxic trophoblast. These data support a functional role for GCM1 contributing to constitutively high trophoblast PGF expression and is the first direct evidence of an oxygen-responsive, trophoblast-specific transcription factor contributing to the regulation of PGF expression.