The mechanism involved in the regulation of transferrin receptor (TfR) expression during phorbol-ester-induced HL-60 cell differentiation was investigated. The mRNA of the TfR was constitutively expressed in proliferating HL-60 cells. Treatment of the cells with phorbol 12-myristate 13-acetate (PMA) for 24 h resulted in a gradual decrease in the expression of the TfR mRNA. Nuclear run-on assays revealed that the transcription of the TfR gene was inhibited by prior treatment of cells with PMA. The effect of PMA on the binding of nuclear proteins to the TfR gene promoter region was then investigated. Based on sequence similarity and previous footprinting data, the promoter region of the TfR gene seems to contain a sequence like that of the phorbol-ester-responsive element (TRE). Our results showed that the binding of nuclear extracts to the TfR gene promoter region containing the TRE-like sequence was increased in PMA-treated cells. This binding activity could be abolished by prior incubation of the nuclear extracts with a synthetic oligonucleotide containing the consensus TRE sequence. In vitro transcription assays revealed that prior incubation of the nuclear extracts of PMA-treated cells with excess consensus TRE oligonucleotide enhanced the gene transcription driven by the TfR gene promoter. These findings suggest that the TRE-like element may play a role in the inhibition of TfR gene transcription.