Two thioredoxin genes from the yeast Saccharomyces cerevisiae were cloned using synthetic oligonucleotide probes. The DNA sequences of the two genes were found to be 74% identical. The two genes, designated TRX1 and TRX2, were mutagenized in vitro and used to construct a set of thioredoxin deletion mutants. The loss of either thioredoxin gene alone has no effect on cell growth or morphology. However, the simultaneous deletion of both thioredoxin genes profoundly affects the cell cycle. S phase is 3-fold longer, and G1 is virtually absent. In addition, the thioredoxin double mutant shows a 33% increase in generation time, a significant increase in cell size, and a greater proportion of large budded cells. The results suggest that in the absence of TRX1 and TRX2, a slow rate of DNA replication inhibits the normal progress of cellular reproduction. Surprisingly, the loss of both thioredoxins also leads to methionine auxotrophy. Thus yeast glutaredoxin is unable to substitute for thioredoxin in sulfate assimilation. As a first step in studying the cell cycle control mechanisms that respond to the thioredoxin deficiency, it was shown that cell viability does not require the function of RAD9, a known cell cycle checkpoint.