Cells derived from a transplantable mouse adrenal cortical tumor maintain their differentiated function in vitro and secrete steroids in response to ACTH and other stimulatory agents. The cell line has been widely employed for various biochemical investigations but there have been few attempts to correlate this work with morphologic data. This communication describes the electron microscopic appearance of the tumor transplant in vivo and primary cultures derived from it at various intervals after the cells are placed in culture. Tumor cells in vivo bear considerable resemblance to normal adult mouse adrenal cortical cells. Organelles generally considered to be directly involved in steroid biosynthesis (mitochondria, smooth endoplasmic reticulum and lipid droplets) are not drastically altered. Certain modifications of the vasculature and cell membrane, seemingly related to steroidogenesis, are present in both the tumor and normal adrenal cortex. Within 2 days after the tumor cells are introduced to culture, their cytoplasm assumes a more simplified appearance. Smooth endoplasmic reticulum is less conspicuous and free ribosomes and polysomes are very abundant. Mitchondrial inner membranes are reorganized from a saccular arrangement in the cells in vivo into distinct lamellar cristae. The tumor cells now resemble undifferentiated embryonic adrenal cells, or cultured adrenal cells from various mammalian sources which have dedifferentiated in the absence of ACTH. In their morphologically unspecialized state, the normal cells are incapable of functional responses to ACTH. In contrast, the cultured, dedifferentiated tumor cells respond within minutes to this hormone and can demonstrate 5-20 fold increases in their basal steroid output. These data suggest that substantial steroidogenic activity can occur although the characteristic appearance of adrenal mitochondria is absent.