The relative physical significance of the two pathways of homocysteine biosynthesis in plants (transsulfuration and direct sulfhydration) is being determined by following the patterns of assimilation of 35SO4 ion into sulfur amino acids. Chlorella growing under steady state conditions with limiting sulfate was found to use predominantly the transsulfuration pathway, with a maximum of only 5 percent of the total homocysteine synthesized by direct sulfhydration. The transsulfuration pathway was shown to operate also in a higher plant (Lemna) growing under steady state conditions with a low concentration of sulfate. Experiments are now under way to quantitate the relative contribution of the two pathways in this plant. Crude extracts of plants have previously been shown to catalyze the enzymic synthesis of homocysteine by direct sulfhydration. This system has been used as a sensitive probe to determine the chemical nature and in vivo concentration of the physiologically important alpha-aminobutyryl donor in the direct sulfhydration pathway. O-Phosphohomoserine was found to be the predominant, perhaps the exclusive, alpha-aminobutyryl donor in such phylogenetically diverse plants as Chlorella and Lemna. The use of O-phosphohomoserine in the sulfhydrase reaction, and as shown previously also for cystathionine synthesis, is unique for green plants.