Adrenocortical steroidogenesis is regulated in addition to a central regulation via the hypothalamus-pituitary-adrenal axis by intra-adrenal mechanisms involving the adrenal medulla. We could previously show that adrenocortical steroidogenesis is stimulated by co-culturing bovine adrenocortical cells with medullary chromaffin cells. This stimulation was due to soluble factors released from the chromaffin cells under basal, unstimulated conditions and involved the increased expression of P450 enzymes, StAR and de novo protein synthesis. In the present study we analyzed the differential regulation of the three cortical zones and characterized secretagogues involved in this stimulation. While cortisol and androstenedione release were increased 10 fold by incubation with chromaffin cell-conditioned medium, aldosterone secretion was not influenced. 80% of the stimulation proved to be due to adrenomedullary epinephrine, norepinephrine, ACTH, PACAP and PG-dependent mechanisms. Other adrenomedullary secretory products, serotonin, Met-enkephalin, Leu-enkephalin, galanin, CGRP, substance P, VIP or NPY did not stimulate steroidogenesis in this system. Our data show that adrenomedullary cells differentially regulate the three adrenocortical zones. This stimulation predominantly depended on epinephrine, norepinephrine, PACAP, and ACTH released from the chromaffin cells and prostaglandin-dependent mechanisms such as interleukin-1.