Steroidogenesis by zona reticularis (ZR) and zona fasciculata (ZF) cells from guinea-pig, macaque and human adrenals has been examined to establish the relative importance of the two cell types in the production of adrenal androgens. Microdissection and equilibrium density sedimentation of enzymically dispersed cells on Percoll gradients have been used to separate the two cell types with varying degrees of purity. Outputs of androstenedione and cortisol were measured for all the species studied and dehydroepiandrosterone sulphate (DHAS) for human adrenal cells. In all three species, the ZF cells produced more cortisol than the ZR cells. This production was stimulated to a greater extent in the ZF than the ZR cells in the guinea-pig, but to a similar extent in the macaque and human cells. Outputs of androstenedione were greater for ZF cells than ZR cells in the guinea-pig both basally and in response to an ACTH stimulus but basal production of this androgen was slightly greater from the ZR than the ZF in tissue incubations (1.6-fold), although cortisol output was still higher for ZF tissue by a factor of 2. Examination of androstenedione/cortisol ratios for all the species, both tissue and cells and in the presence or absence of ACTH, demonstrated clearly the inherent bias for ZR cells towards androgen synthesis by the fact that this ratio was always higher for ZR than for ZF cells or tissue. The most marked difference in this ratio was found in the human cells comparing the purest ZR and the purest ZF cell preparations even though the highest estimated purity of either cell type was only 80%. The difference in the ratio DHAS/cortisol was even greater comparing these human ZR and ZF cell preparations, ranging from 18- to 44-fold basally and in response to ACTH. The preliminary experiment on human adrenal cells indicates that the ZR cells are much more important than the ZF cells in the production of androgens even in the presence of ACTH, in contrast to the other species studied.