The development of mouse pronuclear-stage embryos in media containing various concentrations of thioredoxin was monitored and the influence of antithioredoxin immunoglobulin G (IgG) and heat-treated thioredoxin on the thioredoxin-induced effects was evaluated. A significant increase in the number of four-cell embryos (76.3%) and blastocysts (37.3%) was observed when embryos were cultured in the medium containing 50 micrograms thioredoxin ml-1 compared with the rates (55.8 and 3.8%, respectively) in the basic medium. The number of blastocysts increased significantly to a maximum of 70.2% at 500 micrograms ml-1. The biological activity of thioredoxin was evident after dialysis, but was markedly impaired by the addition of anti-thioredoxin IgG to the culture medium. Treatment at 60 degrees C for 5 min did not affect the enzymatic and biological activity of thioredoxin. More severe heat treatment (121 degrees C for 30 min) attenuated the enzymatic activity to 40% of its initial value and reduced the biological activity (number of blastocysts, from 77.8 to 51.6%). These results indicate that the effect of thioredoxin on the two-cell block is due to the thioredoxin molecule itself, and suggest that disulfide formation within or between proteins resulting from oxidative stress is one of the major causes of the two-cell block.