We have previously shown that non-digestible saccharides (NDS) stimulate intestinal Ca absorption via tight junctions. However, the cellular mechanisms activated by the NDS are not yet known. We investigated the effects of four NDS, difructose anhydride (DFA) III, DFAIV, fructo-oligosaccharides, and maltitol, on intracellular Ca signalling in isolated rat small-intestinal enterocytes. The changes in intracellular Ca(2+) concentration were measured before and after the addition of capric acid (7.5 or 15 mmol/l, a positive control), glycerol, or each NDS (1 or 10 mmol/l) to fura-2-loaded enterocytes. Treatment with capric acid or each NDS caused an immediate and dose-dependent rise in intracellular Ca(2+) concentration. Mechanical and osmotic stimulation achieved by adding glycerol had no effect on intracellular Ca(2+) concentration. The intracellular Ca(2+) concentration in enterocytes treated with DFAIII and fructo-oligosaccharides reached a peak level at about 30 s after stimulation, but those treated with DFAIV and maltitol showed further increases after the initial rapid rise. The maximum change in intracellular Ca(2+) concentration obtained by the application of maltitol was higher than that of DFAIII at 10 mmol/l. These findings suggest that each of the four NDS directly stimulates rat enterocytes, and increases intracellular Ca(2+) concentration. Thus, molecular structure may be more important than the size of the NDS in the induction of Ca signalling in the cells.