We have confirmed and characterised structurally the enzyme conformational changes deduced from the preceding thermodynamic analysis of the adrenal mitochondrial cholesterol-side-chain-cleaving cytochrome P450 spin-state equilibrium. The spin-transition kinetics following rapid pH jumps were multiphasic in aqueous buffer and biphasic in the presence of 35% ethylene glycol. The activation energy between -2 degrees C and 30 degrees C of both phases was exceptionally high (Ea = 147 kJ.mol-1), suggesting the involvement of large-scale conformational changes. The pH and temperature effects on the CD spectrum show that the enzyme is in equilibrium between at least two conformations which are predicted by the thermodynamic model, but which are not directly correlated to the spin state. The CD changes between 260 nm and 280 nm indicate that the conformation prevailing at high temperatures is characterised by a decreased polarity of the tyrosine environments; the changes between 200 nm and 250 nm suggest furthermore a 4% decreased protein helical content.