The role of free calcium as a crucial intracellular messenger in the stimulation of aldosterone biosynthesis by various agonists is well established. Using electropermeabilized or Ca²⁺-clamped adrenal zona glomerulosa (ZG) cells, we have previously shown that Ca²⁺ entry into the mitochondrial matrix is required for the activation of steroidogenesis. We now describe the use of various strategies to answer the following questions: 1. Which pathway does Ca²⁺ follow before triggering steroidogenesis? 2. Which step of steroidogenesis is under the control of Ca²⁺? The first approach combined the patch-clamp method, in the perforated patch configuration, with microfluorimetry of Ca²⁺; in the second approach, ZG cells were transiently transfected with a chimeric cDNA encoding for the calcium-sensitive photoprotein aequorin linked to a mitochondrial targeting presequence; in a third approach, ZG mitochondria were isolated and fractionated into outer membranes, contact sites and inner membranes and the effect of prior exposure of the ZG cells to a physiologically elevated intracellular calcium concentration or to angiotensin II (Ang II) on cholesterol content was then examined in those three mitochondrial fractions. The results of these combined approaches allow us to propose the following scheme: The source of calcium which is predominantly responsible for mediating the steroidogenic effect of potassium appears to be funneled through the T-type calcium channels to close proximity of the mitochondria. This signal, as well as that triggered by Ang II, appears to be relayed within the mitochondrial matrix. This rise of mitochondrial calcium is associated with a transfer of free cholesterol from the outer to the inner mitochondrial membrane, via the contact sites. Thus the main role of the calcium messenger is to promote intramitochondrial cholesterol transfer and supply to the P450_SCC enzyme.