Glycoprotein IIb (GPIIb) is an early and specific marker of the megakaryocytic lineage. We have previously shown that a fragment extending 643 base pairs upstream the transcription start site of the human GPIIb promoter was able to control the tissue-specific expression of the CAT gene in transfection experiments. Four potential GATA-binding sites, located at positions -463, -376, -243, and -54 are present within this fragment. Gel shift analysis revealed that nuclear extracts from the erythroleukemic cell line HEL contain a DNA-binding protein that recognizes these GATA sites. Using an antiserum raised to an hydrophilic region of the transcription factor GATA-1, the HEL GATA-binding protein was found to be GATA-1. Point mutations of the different GATA sites indicated that they did not equally contribute to GPIIb promoter activity. The -463 GATA motif located in an enhancer region is essential for full transcription activity and was found to be dominant upon the other GATA motifs. When this site is mutated, the -54 GATA site appears to be essential for the remaining CAT activity. These results indicate that the transcription factor GATA-1 plays an important role in the regulation of the transcription of the megakaryocyte specific GPIIb gene.