The substoichiometric isotope dilution analysis for manganese(II) in a synergistic extraction system of a chelating agent and a neutral ligand is described. The substoichiometric extarction is based on a substoichiometric amount of 2-thenoyltrifluoroacetone and an excess of 1,10-phenanthroline. The recommended condition and the reproducibility of the present system were examined. The present method was applied for NBS-SRM tomato leaves and NIES-SRM chlorella, and very good results with high accuracy and precision were demonstrated.