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 (tau), the chi2-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 (tau) 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 micromol/l) on the insulin response in vitro, the phase-responses support the contention that pancreatic beta cells may be targets for melatonin action.