Most human cancers express telomerase but its activity is highly variable and regulated by complex mechanisms. Recently, we have proposed that Ets proteins may be important for regulation of telomerase activity in leukemic cells. Here we provide further evidence for the role of Ets family members and related Id proteins in telomerase regulation and characterize the underlying molecular mechanisms. By using PCR-based and gel shift assays we demonstrated specific binding to a core hTERT promoter of Ets2, Fli1, Id2, c-Myc, Mad1, and Sp1 in lysates from subclones of U937 cells. Further analysis of binding of purified proteins and various mutants of the hTERT promoter suggested the existence of a trimolecular Ets-Id2-DNA complex, and Ets inhibitory activity mediated by c-Myc and the Ets binding site on the core hTERT promoter at -293 bp from the transcription initiation site as well as a positive Ets regulatory effect mediate through another Ets binding site at -36 bp. This analysis provided evidence for the existence of negative and positive Ets regulatory site and suggested a complex interplay between Ets/Id family members and c-Myc that may be an important determinant of the diversity of telomerase activity in leukemia and other cancers.