Stress and cortisol are known to have negative effects on vitellogenesis in oviparous species. This provides a physiological context in which to explore in more detail the molecular mechanisms involved in transcriptional interferences between two steroids receptors, the estradiol receptor (ER) and the glucocorticoid receptor (GR). We have previously shown that the cortisol inhibitory effect on rainbow trout (rt) vitellogenesis is the result of a repression of the estradiol-induced ER-positive autoregulation by activated GR. In the present study, we demonstrate that the GR repression involves a proximal region of the rtER promoter that is unable to bind GR. This inhibition is counteracted in part by the orphan receptor COUP-TF1 that has been previously shown to cooperate with ERs on the same promoter. A detailed analysis allowed us to identify a C/EBPbeta-like protein that is implicated in both the maximal stimulatory effect of estradiol and the GR repression. Indeed, GR, through its DNA-binding domain, suppresses the binding of C/EBPbeta on the rtER promoter by protein-protein interactions and thereby prevents the enhancer effect of this transcription factor.