A cell-free protein synthesis in rabbit reticulocyte lysates stops within 2 h, which limits the productivity of the cell-free system. Here we show that the accumulation of a translational inhibitor liberated from the post-ribosomal supernatant during the reaction time is responsible for the halt in the translational reaction. Since EGTA was found to suppress the effect of the inhibitor and the absorbance of a Ca2+ indicator was observed to increase, Ca2+ is suggested to be the inhibitor that accumulates in reticulocyte lysates during the cell-free protein synthesis reaction.