Raffinose family oligosaccharides (RFOs) fulfil multiple functions in plants. In seeds, they possibly protect cellular structures during desiccation and constitute carbon reserves for early germination. Their biosynthesis proceeds by the transfer of galactose units from galactinol to sucrose. Galactinol synthase (GolS), which mediates the synthesis of galactinol from myo-inositol and UDP-galactose, has been proposed to be the key enzyme of the pathway. However, no significant relationship was detected between the extractable GolS activity and the amount of RFOs in seeds from seven pea (Pisum sativum L.) genotypes selected for high variation in RFO content. Instead, a highly significant correlation was found between the levels of myo-inositol and RFOs. Moderately strong relationships were also found between sucrose and RFO content as well as between myo-inositol and galactinol. Further evidence for a key role of myo-inositol for the synthesis of galactinol was obtained by feeding exogenous myo-inositol to intact pea seeds and by the analysis of four barley (Hordeum vulgare L.) low phytic acid mutants. In seeds of three of these mutants, the reduced demand for myo-inositol for the synthesis of phytic acid (myo-inositol 1,2,3,4,5,6-hexakisphosphate) was associated with an increased level in myo-inositol. The mutants seeds also contained more galactinol than wild-type seeds. The results suggest that the extent of RFO accumulation is controlled by the levels of the initial substrates, myo-inositol and sucrose, rather than by GolS activity alone.