Long-term ACTH-stimulation of steroidogenesis in the rat adrenal cortex results in time-dependent increases in the surface area per cell of smooth endoplasmic reticulum and mitochondrial cristae. As the morphological responses to short-term ACTH stimulation have not been described, we undertook morphometric analyses of the effects of acute (10 min) ACTH stimulation of rat adrenocortical cells in vivo as they may be expressed in the mitochondria and the smooth endoplasmic reticulum. Six young male Wistar rats were allocated to each of four groups: 1. normal controls; 2. ACTH-treated normal rats; 3. Dexamethasone-inhibited; 4. ACTH-treated Dexamethasone-inhibited. As judged by the radio-immunoassay of trunk blood, levels of ACTH, 11-deoxycorticosterone and corticosterone were appropriate to the treatment state. ACTH activation resulted in no changes in the smooth endoplasmic reticulum; but the mitochondrial inter-membrane space was significantly increased over that of the contrasted pair. The inter-membrane space in the dexamethasone-inhibited rats was significantly less than that of all other groups. No responses to ACTH-activation were shown by the intra-cristal or matrix volumes of the mitochondria. The increased inter-membrane space appears to be caused by a decrease in the surface area of the inner mitochondrial membrane. The significance of these intra-mitochondrial changes to the rate-limiting step of steroidogenesis is discussed.