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Mixtures of zona fasciculata (ZF) and zona reticularis (ZR) cells, obtained by enzyme dispersion of decapsulated guinea-pig adrenal glands, were separated either by unit gravity sedimentation or by equilibrium density sedimentation. There was no evidence of deleterious effects on ultrastructural integrity or the ability of cells to respond to (1–24)ACTH (Synacthen) after either separation technique. Unit gravity sedimentation gave one fraction in which 90% of the cells were from the ZR and another fraction in which 70% of the cells were from the ZF. Equilibrium density sedimentation of cell mixtures on Percoll gradients gave fractions containing either 90% pure ZR or 95% pure ZF cells.

Cortisol, 11-deoxycortisol, corticosterone, deoxycorticosterone, 11 β-hydroxyandrostenedione and androstenedione were all formed from [¹⁴C]pregnenolone on incubation with purified preparations of both types of cell. No product was seen to be unique to either cell type although ZR cells appeared deficient in 11β-hydroxylase activity relative to ZF cells. The ratio of androstenedione to cortisol (formed either from labelled pregnenolone or from endogenous precursors) was higher for ZR cells than for ZF cells. When the purer cells obtained by equilibrium density sedimentation were studied, it was found that (1–24)ACTH stimulated greater steroid production (both androstenedione and cortisol) by the ZF cells compared with the ZR cells.

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