The human ETS1 gene promoter contains binding sites for transcription factors AP1, AP2 and ETS. These factors have been previously shown to be positive regulators for the expression of the ETS1 gene. In our previous report it was inferred that the ETS1 gene promoter also contains negative regulatory elements that might balance and prevent the overexpression of the ETS1 gene product. The studies reported here show that the ETS1 gene promoter also contains binding site motifs for transcription factors PEA3 and OCT. By subjecting deletion constructs of the ETS1 gene promoter to functional analysis, two negative regulatory elements (NREs) were located: NRE1 was mapped to 230 nt at the 5' end of the promoter, and NRE2 was mapped to 350 nt located between the second OCT motif and a 120-base sequence downstream from the SacI site. These NREs can also reduce the activity of a heterogenous promoter. Gel mobility-shift assays showed that nuclear extracts from B-lymphoid (Daudi), T-lymphoid (HPB) and erthyromyeloid (K562) cells each form one major and several minor complexes with NRE1 and NRE2. The nuclear extract from promyelocytic (HL60) cells does not form complexes with either NRE1 or NRE2. The identification of NREs in the ETS1 gene promoter suggests that these elements play an important role in regulating the ETS1 gene expression in hematopoietic cells.