To gain insight into the evolution of the methionine biosynthesis pathway, in vivo complementation tests were performed. The substrate specificity of three enzymes that intrinsically use different homoserine-esterified substrates and have different sulfur assimilation pathways was examined: two cystathionine gamma-synthases (the Escherichia coli enzyme that naturally utilizes O-succinylhomoserine [OSH]) and the Arabidopsis thaliana enzyme that naturally exploits O-phosphohomoserine [OPH]. Both of these act through the transsulfuration pathway. The third enzyme investigated was O-acetylhomoserine (OAH) sulfhydrylase of Leptospira meyeri, representing the enzyme that utilizes OAH and operates through the direct sulfhydrylation pathway. All the three enzymes were able to utilize OSH and OAH as substrates, with different degrees of efficiency, but only the plant enzyme was able to utilize OPH as a substrate. In addition to their inherent activity in the transsulfuration pathway, the two cystathionine gamma-synthases were also capable of acting in the direct sulfhydrylation pathway. Based on the phylogenic tree and the results of the complementation tests, we suggest that the ancestral gene was able to act as OAH or OSH sulfhydrylase. In some bacteria and plants, this ancient enzyme most probably evolved into a cystathionine gamma-synthase, thereby maintaining the ability to utilize various homoserine-esterified substrates, as well as various sulfur sources, and thus keeping the multisubstrate specificity of its ancestor. In some organisms, this ancestral gene probably underwent a duplication event, which resulted in a cystathionine gamma-synthase and a separate OAH or OSH sulfhydrylase. This led to the development of two parallel pathways of methionine biosynthesis, transsulfuration and direct sulfhydrylation, in these organisms. Although both pathways exist in several organisms, most seem to favor a single specific pathway for methionine biosynthesis in vivo.