A superfusion system was developed in which secretion of cortisol from the interrenal tissue of Sarotherodon aureus could be stimulated by ACTH. Head kidneys from four fish were cut into fragments and superfused with Eagle's basal medium containing 4 mM-NaHCO3 and bovine serum albumin (50 mg/100 ml). The superfused medium was collected every 15 min before stimulation and every 5 min thereafter. Cortisol was measured in the medium by radioimmunoassay. The rate of cortisol secretion increased considerably after a 5 min pulse of 0.1 mu. porcine ACTH/ml, reaching a peak of 11.64 ± 2.40 (S.E.M.) ng/min (n = 5) after 20 min. In the subsequent 35 min the secretion rate decreased to a level of 0.49 ± 0.21 ng/min which was within the range of the baseline (0.2-0.7 ng/min). The amount of cortisol secreted by the superfused tissue in response to the corticotrophin, calculated from the area under the peak, was dose-dependent at the range of 0.06-273 mu. ACTH. Cortisol secretion in this system could also be stimulated by a crude pituitary extract from the same fish. Using the dose-response line, the adrenocorticotrophic activity in the pituitary gland of S. aureus was estimated as 0.2 i.u./g or 1.87 mu. per gland, in porcine ACTH equivalents. It was possible to substitute ACTH with dibutyrylic cyclic AMP. The response of the interrenal tissue, i.e. the increase in secretion of cortisol, was dose-dependent at the range of 2-20 mmol/l. The response of the superfused interrenal tissue to ACTH could be extended by the addition of the synthetic phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine (0.1 mmol/l), to the medium. These results indicated that the stimulation of cortisol secretion from the interrenal tissue of this fish by ACTH is, most probably, mediated by cyclic AMP as a second messenger.