It is generally believed that the initiation of insulin secretion by nutrient stimuli necessitates the generation of metabolic coupling factors, leading to membrane depolarization and the gating of voltage‐sensitive Ca2+ channels. To establish this sequence of events, the kinetics of endogenous fluorescence of reduced pyridine nucleotides [NAD(P)H], reflecting nutrient metabolism, were compared to those of cytosolic calcium ([Ca2+]i) rises in single cultured rat islet beta‐cells. In preliminary experiments, the loss of quinacrine fluorescence from prelabelled cells was used as an indicator of secretion. This dye is concentrated in the acidic insulin‐containing secretory granules. Both glucose and 2‐ketoisocaproate (KIC) raised [Ca2+]i in a dose‐dependent manner. There was marked cellular heterogeneity in the [Ca2+]i response patterns. The two nutrient stimuli also increased NAD(P)H fluorescence, again showing cell‐to‐cell variations. In combined experiments, where the two parameters were measured in the same cell, the elevation of the NAD(P)H fluorescence preceded the rise in [Ca2+]i, confirming the statistical evaluation performed on separate cells. The application of two consecutive glucose challenges revealed coordinated changes in [Ca2+]i and NAD(P)H fluorescence. Finally, quinacrine secretion was stimulated by two nutrients with onset times similar to those recorded for [Ca2+]i elevations. These results clearly demonstrate that increased metabolism occurs during the lag period preceding Ca2+ influx via voltage‐sensitive Ca2+ channels, a prerequisite for the triggering of insulin secretion by nutrient stimuli.