A method has been developed for the qualitative and quantitative analysis of complex isotopic mixtures of tritium-labelled amino acids and peptides by using high resolution(3)H NMR spectroscopy at 266.8 MHz. Determined were tritium distribution in alanine, glycine, tryptophan and 4-hydroxyproline amino acids, as well as in glycine and valine residues of peptides. Approaches have been worked out for the determination of spin coupling constants and isotope chemical shifts for the strongly coupled nonequivalent atoms of the methylene groups.