We tested the hypothesis that the circadian clock modulates Ca2+-based signalling pathways, using low-temperature (LT)-induced Ca2+ signals. We investigated the relationship between diurnal and circadian modulation of LT-induced increases in cytosolic-free calcium ([Ca2+](cyt)), and regulation of [Ca2+](cyt)-dependent outputs of the LT-signalling network (RD29A transcript abundance and stomatal closure). We measured [Ca2+](cyt) non-invasively using aequorin, and targeted aequorin to the guard cell using a guard cell-specific GAL4-green fluorescent protein enhancer trap line. LT caused transient increases in whole plant and guard cell [Ca2+](cyt). In guard cells, the LT-induced [Ca2+](cyt) elevation preceded stomatal closure. In whole plants, the magnitude of LT-induced [Ca2+](cyt) transients, measured from the entire plant or specifically the guard cell, varied with the time of day: LT-induced [Ca2+](cyt) transients were significantly higher during the mid-photoperiod than at the beginning or end. Diurnal variation in LT-induced guard cell [Ca2+](cyt) increases was not correlated to diurnal variation in LT-induced stomatal closure. There was circadian modulation of LT-induced whole plant [Ca2+](cyt) increases, which were correlated to the circadian pattern of RD29A induction. In order to understand the significance of LT-induced [Ca2+](cyt) increases, we used a computer simulation to demonstrate that, in guard cells, LT-induced [Ca2+](cyt) increases measured from a population of cells are likely to represent the summation of cold-induced single-cell [Ca2+](cyt) oscillations.