The guinea pig adrenal cortex is grossly composed of two regions: an outer, yellow zone and an inner, brown zone. These zones, which represent 33% and 66% of the total adrenocortical volume, respectively, can be separated by blunt dissection. It has been previously reported that specific pregnenolone and pregnenolone sulfate binding proteins are present in the high speed supernatant fraction (cytosol) prepared from the whole adrenal cortex of the guinea pig. However, when cytosol was prepared from the separate outer and inner cortical zones, it was found that the steroid-binding proteins were concentrated in the inner zone. This correlated with the level of pregnenolone which was significantly greater in the cytosol of the inner zone where greater than 50% was found to be bound. In contrast, the concentration of cortisol was 30 times greater in the cytosol of the outer cortical zone and less than 4% was found to be bound. These data suggest that cortisol is produced primarily in the outer cortical zone, a region which comprises only one-third of the total cortical volume. On the other hand, the coexistence of pregnenolone and its binding protein in the inner cortical zone, a region which comprises two-thirds or the greatest cortical volume, indicates a different functional status for this zone. The exact hormonal control of these two vastly different regions (chromatically, morphologically, and functionally) remains to be determined. It is speculated that the inner cortical zone of the adult guinea pig adrenal is the counterpart of the fetal cortex which did not involute.