Messenger RNA was isolated from young excised tomato leaves, induced to accumulate proteinase Inhibitors I and II with the proteinase inhibitor inducing factor (PIIF), and translated in vitro in a rabbit reticulocyte lysate system. Translatable messenger RNAs specific for Inhibitors I and II were present in PIIF-induced leaves but were not present without PIIF induction. The nascent in vitro-synthesized inhibitors migrated with an apparent molecular weight 2,000 to 3,000 daltons larger than that of the two inhibitors isolated from leaves. The molecular weights of the preinhibitors were identical whether translated from mRNA from PIIF-induced leaves or translated from mRNA isolated from wounded leaves. Incubation of excised PIIF-induced plants in CO2-free air doubled the rate of in vivo synthesis of Inhibitor I over that in normal air (Ryan CA 1977 Biochem Biophys Res Commun 77: 1004-1008) but did not affect the rate of in vivo Inhibitor II accumulation. The rate of incorporation of 35SO42− into soluble proteins was 70% less when leaves were incubated in CO2-free air rather than normal air. Messenger RNA isolated from PIIF-induced plants incubated in the presence or absence of CO2 was translated in vitro. The amount of in vitro-translatable mRNA present for each inhibitor (per microgram total mRNA) was the same in leaves incubated in either atmosphere. Therefore, the increased rate of synthesis and accumulation of Inhibitor I in a CO2-free atmosphere does not appear to result from an increased level of mRNA but appears to be controlled at a posttranscriptional level.