An Escherichia coli S-30 DNA directed protein synthesis system was used to study the effect of homocysteine on the in vitro expression of the metE, metH and metR genes. In the presence of purified MetR protein, which is known to regulate the expression of these genes, homocysteine activates metE expression and inhibits both metR and metH expression. These findings support the recent in vivo results of Urbanowski, M.L. and Stauffer, G.V. (1989), J. Bacteriol. 171, 3277-3281.