We have studied the in vivo expression of steroidogenic acute regulatory protein (StAR) in adrenals of control, ACTH-treated, and Na⁺-restricted rats. Indirect immunofluorescence by microscopy revealed the presence of StAR in the zonae glomerulosa (ZG) and fasciculata-reticularis (ZFR). An increased signal was observed in the ZG and zona fasciculata, 5 h after ACTH injection; a few cells of the medulla were also positive. Immunogold electron microscopy showed that StAR was mainly located over mitochondria (MT). By immunoblotting, a major 29-kDa and other minor StAR bands migrating between 30 and 39 kDa were increased 5 h after ACTH treatment but remained unchanged after 1 h. By two-dimensional-PAGE, four StAR species were revealed in homogenates of control ZG, and their intensity was increased 5 h after ACTH treatment but not after 1 h. Also, additional acidic species were seen 5 h after treatment. Other bands with basic isoelectric point were revealed between 29 and 37 kDa. Analyses on whole gland MT and supernatant (SN) revealed four bands in the control SN and five in ACTH SN; the intensity of one band was increased, and that of another one was decreased, in SN of treated rats. ACTH treatment resulted in the localization of many low-isoelectric point StARs in MT. After two-dimensional-PAGE, differences were found in the mobility of some StAR species in the ZG between controls and Na⁺-restricted rats. In MT, four bands were revealed in the ZG preparations of Na⁺-restricted and two bands in controls. Four bands were revealed in the ZG SNs of control and Na⁺-restricted rats; an additional band was observed only in the SN of treated animals, whereas the intensity of another band decreased. Na⁺ restriction did not affect StAR in the ZFR. In conclusion, StAR was present in the rat adrenal cortex ZG and ZFR and was mainly located in MT. StAR expression was inducible in the ZG and the ZF by ACTH, resulting in the formation of many StAR acidic species; interestingly, such changes were detectable 5 h, but not 1 h, after ACTH administration, suggesting that steroidogenesis stimulation by StAR might occur mainly outside MT. Although less spectacular than for ACTH, Na⁺ restriction also affected StAR expression in the ZG but not in the ZFR, by increasing two mitochondrial and one SN species, implying that StAR is involved in the mechanism of action of Na⁺ restriction in promoting aldosterone formation. These results suggest that differential processing and/or changes in phosphorylation may occur in vivo upon ACTH treatment and Na⁺ restriction. We hypothesize that modification of a relatively small quantity of StAR, mainly located outside MT, is necessary to increase adrenal steroidogenesis challenged either by ACTH or Na⁺ restriction.