While there is strong evidence that circadian rhythms are generated in the mammalian suprachiasmatic nucleus (SCN) [1,2] and in the avian pineal gland [3,4], determinations of circadian rhythms in other isolated organs or cells are rare. Only few in vitro investigations of insect tissues or mammalian cells or organs such as intestine, adrenals [5±7], heart cell networks and liver cells [8,9] have shown the existence of circadian rhythms in culture [10,11]. In addition it has been shown that cultured mammalian retina possesses a genetically programmed circadian oscillator which regulates the synthesis of melatonin in this tissue. These observations suggest that all vertebrate photoreceptive structures synthesize melatonin under the control of circadian oscillators [12]. A circadian rhythm is an oscillation with a period duration of approximately 24 h (range 20 to 28 h). Ultradian rhythms are oscillations with periods of less than 20 h.Various investigators have postulated oscillations of insulin secretion in a range of seconds [13] or periods between 9 and 14 min under both in vivo and in vitro conditions [14] or clonal pancreatic beta cells with periods of 5 to 8 min superimposed on 15 to Diabetologia (1998) Summary This study aims to analyse a circadian rhythm of insulin secretion from isolated rat pancreatic islets in vitro and its potential modulation by melatonin, the concentrations of which change in vivo inversely to that of insulin. The circadian rhythm was evaluated in a perifusion system, adapted to the specific conditions of pancreatic islets. To determine rhythmicity of insulin secretion, 30-min fractions were collected continuously for investigative periods of 44 to 112 h. Insulin secretion in 10 experiments was analysed by using the MacAnova-program for period length (t), the c 2 -periodogram for test of significance (p < 0.001), and additionally the empirical cosine adaptation for amplitude and goodness-of-fit. Thereby a circadian pattern was observed with periods (t) between 21.8 and 26.2 h. The period duration (mean ± SEM) was 23.59 ± 0.503 h, the overall mean insulin release 1038 ± 13 pmol/l and the mean amplitude 88 ± 17 pmol/l. Adding melatonin (10 nmol/l, t = 2 h) as a hormonal Zeitgeber during analysis of circadian insulin secretion phase-response studies show phase-shifts with approximately 9 h phase advance. Thereafter the circadian period was maintained, while the amplitude was enhanced. From this it is concluded that an endogenous circadian oscillator is located within the pancreatic islets of the rat that regulates circadian insulin secretion of the insulin-producing beta cells. The pacemaker is remarkably stable, because its periodicity is not affected by factors altering insulin secretion. In agreement with inhibitory influences of melatonin (range 0.5 nmol/l to 5 mmol/l) on the insulin response in vitro, the phaseresponses support the contention that pancreatic beta cells may be targets for melatonin action. [Diabetologia (1998)