The human aromatase (hCYP19) gene is controlled by tissue-specific promoters that lie upstream of tissue-specific first exons. Placenta-specific exon I.1 lies 100,000 bp upstream of exon II. Previously, we observed that genomic sequences within 501 bp upstream of exon I.1 mediate placenta-specific expression. In the present study, transgenic mice were created carrying hCYP19I.1_-246:hGH/hGX, hCYP19I.1_-201:hGH and hCYP19I.1_-125:hGH fusion genes to further delineate 5'-flanking sequences within 501 bp of exon I.1 that are required to mediate placenta-specific hCYP19 gene expression. As little as 246 bp of hCYP19 exon I.1 5'-flanking sequence was sufficient to direct placenta-specific expression in transgenic mice. By contrast, transgenes containing 201 bp or 125 bp of exon I.1 5'-flanking DNA were not expressed in mouse placenta. Furthermore, hCYP19I.1_-246:hGX transgene expression was developmentally regulated; expression was observed as early as embryonic day (E) 7.5 in several cells of the trophoblast ectoderm, at E8.5 in some trophoblast giant cells and by E9.5 in giant cells and labyrinthine layer. By contrast, expression of the hCYP19I.1_-501:hGH transgene was first observed at E10.5 and restricted to the labyrinthine layer, which is most analogous to human syncytiotrophoblast. This suggests the presence of regulatory elements between -501 and -246 bp that may bind inhibitory transcription factors expressed in giant cells. These findings from transgenic experiments, together with deletion mapping studies using transfected human placental cells, indicate that the concerted interaction of strong placenta-specific enhancers and silencers within this 501 bp region mediate labyrinthine and syncytiotrophoblast-specific CYP19 gene expression