Thioredoxin from Escherichia coli B and phage T4-infected E. coli B are small hydrogen carrier proteins which in their reduced forms are specific hydrogen donors to E. coli and T4-induced ribonucleotide reductase, respectively. The oxidation-reduction active group of both thioredoxins consists of a single cystine residue which is reduced to sulfhydryl form by NADPH in the presence of E. coli thioredoxin reductase. Reduction of T4 thioredoxin-S2 to thioredoxin-(SH)2 led to a 3-fold increase in the quantum yield of tyrosine fluorescence. By using the spectrofluorimetric properties of T4 thioredoxin and E. coli thioredoxin as markers for their oxidized and reduced forms we have shown that E. coli thioredoxin reductase catalyzed the reaction: (see article) whose equilibrium constant favors formation of E. coli thioredoxin-S2 and T4 thioredoxin-(SH)2. This finding suggests that in the T4-infected cell most of the deoxyribonucleotides required for the viral DNA might be synthesized by the T4-induced ribonucleotide reductase while the host ribonucleotide reductase is inactive due to the shortage of reduced E. coli thioredoxin.