In the rat platelet factor 4 (PF4) promoter, Ets motifs and GATA motifs are located at positions −880, −75 and −135, −30, respectively, and their motifs are found in the promoter region of most megakaryocyte protein genes. In order to investigate how the Ets and GATA motifs affect PF4 promoter activity, we constructed Ets and/or GATA motif mutant genes. A single disruption of either −75Ets, −135GATA, or −30GATA significantly reduced PF4 promoter activity, and double disruptions involving these motifs completely abolished it. Furthermore, gel‐retardation assays revealed that Ets‐1 and GATA‐1 proteins from HEL and MEG‐01 cells bound to the Ets motifs and GATA motifs, respectively. Co‐transfection experiments showed that the overexpression of Ets‐1 and/or GATA‐1 enhanced the expression of the PF4 promoter reporter gene. These effects of Ets‐1 and GATA‐1 on PF4 promoter activity are additive. When HEL cells were treated with dimethylsulfoxide in order to induce differentiation into megakaryocytes, the mRNA level of ets‐1 increased 10‐fold, which might be directly correlated with the significant increase in PF4 mRNA level induced by dimethylsulfoxide. All these results strongly suggest that both Ets‐1 and GATA‐1 play key roles in the positive regulation of PF4 gene expression.