Naturally occurring diabetogenic substance (NDS), isolated from clinical grade human growth hormone (hGH), induces insulin release from isolated pancreatic islets of hypo-physectomized rats in the presence of Krebs–Ringer bicarbonate solution (KRB) containing 2·8 mmol glucose/l. To determine the role of extracellular glucose in insulin release mediated by NDS, islets were perifused with glucose-free KRB containing 200 μg NDS/ml. Under these conditions NDS induced prompt insulin release (284 ± 34 (s.e.m.)% over basal insulin secretion) (P < 0·0005). Islets perifused with 16·7 mmol glucose/l before and during exposure to 200 μg NDS/ml released additional insulin with exposure to NDS (199 ± 28% over basal insulin secretion) (P< 0·0005). Purified intact hGH (200 μg/ml) did not induce insulin release in the presence of 16·7 mmol glucose/l. Mannoheptulose (5 mmol/l) did not inhibit insulin release mediated by NDS but did inhibit insulin release stimulated by 16·7 mmol glucose/l (P< 0·0005). Islets were pre-incubated for 90 min with 200 μg NDS/ml or 200 μg intact hGH/ml KRB and 2·8 mmol glucose/l to determine what effect either protein might have on subsequent glucose-stimulated (16·7 mmol/l) insulin release. Islets pre-incubated with NDS responded with no less insulin release than islets pre-incubated with intact hGH. Naturally occurring diabetogenic substance initiated insulin release in the absence of extracellular glucose, stimulated additional secretion in the presence of stimulatory glucose concentrations and did not inhibit subsequent islet response to glucose. Naturally occurring diabetogenic substance did not depend on glucose phosphorylation to initiate insulin release.