ZNF300 was recently identified as a member of the human KRAB/C(2)H(2) zinc finger protein family. Little is known about the role of ZNF300 in human gene regulation networks. In this study, the DNA-binding property of ZNF300 was further analyzed. We found that the recombinant ZNF300 could bind to the binding site 5'-GCGGGGGCG-3' of Egr1, another member of the KRAB/C(2)H(2) zinc finger protein family. Similarly, recombinant Egr1 also showed a similar binding affinity to the ZNF300 binding site 5'-CTGGGGGCG-3'. Bioinformatics analysis revealed that there is an overlapping ZNF300/Egr1 binding site in the human IL-2Rβ promoter region, which was previously known to be recognized by endogenous Egr1. Electrophoretic mobility shift assays showed that endogenous ZNF300 could also bind to this site. A transient transfection assay revealed that both ZNF300 and Egr1 could transactivate the IL-2Rβ promoter, and that the activation was abrogated by a mutation of residues in the overlapping ZNF300/Egr1 binding site. Co-expression of ZNF300 and Egr1 led to enhanced IL-2Rβ promoter activity. Thus, ZNF300 is likely to be another regulator of the human IL-2Rβ promoter.