An Escherichia coli mutant deficient in cystathionine beta-lyase was found to accumulate a substance detectable by ninhydrin reaction and chloride platinic acid reaction in its cells (rarely in the culture supernatant) when cultured with a limited amount (50-200 mug/ml) of L-methionine to support the growth. The product was released by freezing treatment and isolated by ion-exchange chromatography (cation exchange resin: Daiaion SK1B). It was identified as L-cystathionine by liquid chromatography-mass spectrometry, C-13- and H-1-nuclear magnetic resonance analyses and high-performance liquid chromatography (as its 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate derivative).