To facilitate studies on differentiation of adrenocortical cells and regulation of steroidogenic genes, we established cell lines from adrenals of adult transgenic mice harboring a temperature-sensitive large T-antigen gene of simian virus 40. Adrenal glands of the mice exhibited normal cortical zonation including a functionally undifferentiated cell-layer between the aldosterone-synthesizing zona glomerulosa cells and the corticosterone-synthesizing zona fasciculata cells. At a permissive temperature (33 degrees C), established cell lines AcA201, AcE60 and AcA101 expressed steroidogenic genes encoding steroidogenic factor-1, cholesterol side-chain cleavage P450scc, and steroidogenic acute regulatory protein, which are expressed throughout adrenal cortices and gonads. Genes encoding 3 beta-hydroxysteroid dehydrogenase and steroid 21-hydroxylase P450c21, which catalyze the intermediate steps for syntheses of both aldosterone and corticosterone, were inducible in the three cell lines in temperature- and/or dibutyryl cAMP-dependent manners. Notably, these cell lines displayed distinct expression patterns of the steroid 11 beta-hydroxylase P45011 beta gene responsible for the zone-specific synthesis of corticosterone. AcA201 cells expressed the P45011 beta gene at 33 degrees C, showing the property of the zona fasciculata cells, while AcE60 cells expressed it upon a shift to a nonpermissive temperature (39 degrees C). On the other hand, AcA101 expressed the P45011 beta gene at 39 degrees C synergistically with exposure to dibutyryl cAMP. None of these clones express the zona glomerulosa-specific aldosterone synthase P450aldo gene under the conditions we tested. These results show that AcE60 and AcA101 cells display a pattern of the steroidogenic gene expression similar to that of the undifferentiated cell-layer and are capable of differentiating into the zona fasciculata-like cells in vitro.