The effects of somatostatin-14 (SS-14) and salmon somatostatin-25 (sSS-25) on hepatic glycogenolysis were studied by incubating rainbow trout liver pieces in vitro. Glycogen content in untreated liver pieces cultured for 3 and 5 hr was 28.6 ± 7.6 mg/g fresh wt. and 21.5 ± 6.6 U, respectively. Treatment of liver pieces with either SS-14 or sSS-25 resulted in significant glycogen depletion; sSS-25 appeared more potent in this regard. Equimolar concentrations (10-8 M) of SS-14 or sSS-25 reduced glycogen content to 10.6 ± 1.6 and 2.6 ± 2.2 U, respectively, in liver pieces incubated for 3 hr. Alterations in liver glycogen content were reflected in glucose release into medium. Basal release of glucose into culture medium over the course of a 3-hr incubation was 20 ± 5.6 μmol/g dry wt. Both SS-14 and sSS-25 stimulated a rapid increase (500 and 600%, respectively) in glucose release during the first 0.5 hr of incubation. After 3 hr, SS-14 and sSS-25 stimulated glucose release over basal levels to 116 ± 9.3 and 153 ± 16 U, respectively. Both SS-14 and sSS-25 stimulated glucose release in a dose-dependent manner; ED50 for both peptides was ca. 5 × 10t-8 M. These results indicate that both SS-14 and sSS-25 directly mediate hepatic glycogenolysis in rainbow trout.