The 'substantial equivalence' of three transgenic wheats expressing additional high-molecular-weight subunit genes and the corresponding parental lines (two lines plus a null transformant) was examined using metabolite profiling of samples grown in replicate field trials on two UK sites (Rothamsted, Hertfordshire and Long Ashton, near Bristol) for 3 years. Multivariate comparison of the proton nuclear magnetic resonance spectra of polar metabolites extracted with deuterated methanol-water showed a stronger influence of site and year than of genotype. Nevertheless, some separation between the transgenic and parental lines was observed, notably between the transgenic line B73-6-1 (which had the highest level of transgene expression) and its parental line L88-6. Comparison of the spectra showed that this separation resulted from increased levels of maltose and/or sucrose in this transgenic line, and that differences in free amino acids were also apparent. More detailed studies of the amino acid composition of material grown in 2000 were carried out using gas chromatography-mass spectrometry. The most noticeable difference was that the samples grown at Rothamsted consistently contained larger amounts of acidic amino acids (glutamic, aspartic) and their amides (glutamine, asparagine). In addition, the related lines, L88-6 and B73-6-1, both contained larger amounts of proline and gamma-aminobutyric acid when grown at Long Ashton than at Rothamsted. The results clearly demonstrate that the environment affects the metabolome and that any differences between the control and transgenic lines are generally within the same range as the differences observed between the control lines grown on different sites and in different years.