We established the first adrenocortical tumor cell lines (ATC) with complete zona fasciculata (ZF) cell phenotype from tumors induced in transgenic mice by large T-antigen of simian virus 40 under the control of the aldose reductase-like akr1b7 gene promoter. ATC lines produced high amounts of corticosterone and were responsive to ACTH. All genes that are supportive for glucocorticoid synthesis including cyp21a1 and cyp11b1 were expressed and most of them were transiently up-regulated by ACTH at transcriptional level : stimulation culminated after 3-6 h and returned to basal levels after 24 h. Making advantage of these cells, we have examined the effect of ACTH on DAX-1 and SF-1, two transcription factors known to respectively repress and activate adrenocortical steroidogenesis by acting on common target genes. According to their antagonistic activities, DAX-1 mRNA and protein levels were transiently down-regulated by ACTH whereas those of SF-1 were stimulated, with kinetics paralleling those of steroidogenic genes expression, notably of two known SF-1 target genes, star and akr1b7. This suggests an essential role of SF-1/DAX-1 proteins ratio to achieve proper ACTH control of steroidogenic gene expression in cells derived from ZF. This was confirmed in mice adrenals, where repression of dax-1 gene and concomitant up-regulation of sf-1, star and akr1b7 genes were observed in response to ACTH stimulation. In conclusion, using both unique differentiated cell lines and in vivo approaches, we provide the first evidence that hormonally-induced changes in SF-1/DAX-1 ratio are part of the molecular arsenal of ZF cells to fine tune ACTH responsiveness.