The cytotoxic activities of restrictocin with aminoterminal extensions and specific mutations were investigated using in vivo and in vitro systems. Genes were constructed from the cDNA clone of restrictocin which encode: the native form of restrictocin (including the leader sequence); Met-prorestrictocin, in which a codon for methionine was placed before a putative pro region; Met-mature restrictocin, with a methionine codon prior to the mature form of restrictocin; and three mutated forms of Met-mature restrictocin, E95G, E115G/H136L, and H136L. These constructions were placed under the control of the GAL1 promoter and were transformed into Saccharomyces cerevisiae. Transformants were killed, and a new RNA band formed when any of these genes except those containing the H136L mutation were expressed. Restrictocin protein was detected by immunoblot only in cells expressing the native form of restrictocin and the forms containing the H136L mutation. Native restrictocin, Met-prorestrictocin, and Met-mature restrictocin mRNA were translated in an in vitro system resulting in proteins of the expected molecular weight and inactivation of the translation system. Restrictocin was not inactivated by the presence of the leader sequence and the putative prosequence. Amino acid His136 is putatively in the active site of restrictocin by analogy to ribonuclease U2 and the elimination of toxic effects in the S. cerevisiae expression and in vitro translation systems.