We examined noncooperativity of estrogen action by studying the transcription of estrogen receptor (ER) down-regulation in an ER stably transfected cell line (Rat1 + ER). The time-course of the ER transcription rate following 17beta-estradiol (E2) (10 nM) treatment was measured by nuclear run-on assays. ER transcription rates decreased to 40 +/- 3% within 1 h as compared with no treatment and stayed suppressed to 24 h of E2 treatment. The ER transcription rate decrease was noncooperative when measured at 24 h of E2 treatment. This suggested that initial E2 binding to the ER is also a noncooperative process. The Hill coefficients of E2 binding to ER were near unity with an equilibrium dissociation constant of 1-3 nM when measured after a 1 h incubation. These results show that E2 noncooperatively binds ER and down-regulates transcription of ER noncooperatively in intact Ratl + ER cells.