This study was undertaken to better understand the physiological role of the retinoylation process in steroidogenic tissues. In adrenal gland mitochondria, the retinoylation extent was found equal to that of testes mitochondria but without ATP in the incubation buffer. We pointed out that the endogenous mitochondrial ATP in adrenal glands is much higher than in testes, about 1.3 × 10−2M and 5.2 × 10−8M, respectively. In addition, less CoASH is required for the maximal acylation activity of the retinoyl moiety to protein(s) compared to testes. The fatty acid analysis revealed a different composition of mitochondrial membranes of these two tissues. Among the different values of fatty acids, it is important to note that adrenal glands contain a much higher amount of C18:0 and a much lower amount of C22:5 ω6 and C22:6 ω3 than testes in the mitochondrial membranes. In addition, there were also differences in arachidonic acid (ARA, C20:4 ω6) content between adrenal glands and testes mitochondria. These different values in the fatty acids composition should explain the different extent of the retinoylation process between the two organs.