A sensitive and specific method for measuring cystathionine is described. The method utilizes the ability of a crude enzyme extract from Bacillus subtilis to catalyze the exchange of [¹⁴C]cysteine into cystathionine. The [¹⁴C]cystathionine thus formed is isolated and its radioactivity determined. The exchange reaction was shown to be specific, and proportional to the amount of cystathionine present in the reaction, within the range from 0.025 to 1 nmole. Tissue extracts to be assayed were purified by ion exchange chromatography, electrophoresis, and paper chromatography, to concentrate the cystathionine and to remove interfering compounds. Application of the method to plant tissues showed that cystathionine is a widespread plant constituent. It is present in plants covering a wide phylogenetic range, as well as in various tissues, at concentrations ranging from 0.04 to 2.6 nmole/g wet wt.