Incorporation of [3H]putrescine into proteins was shown to increase markedly in sea urchin eggs upon fertilization. Emetine, an inhibitor of protein synthesis, had no effect on the rate of protein labeling. However, the reaction could be prevented by the addition of 2-[3-(diallylamino)-propionyl]benzothiophene, a noncompetitive inhibitor of transglutaminase, and also by dansylcadaverine, which is a substrate for transglutaminase. The inert N alpha-dimethyl analogue of dansylcadaverine had no influence. Considering the complexity of the incorporation of the [3H]putrescine tracer in this system, it was deemed essential to prove by rigorous analytical methods that the reaction was, indeed, consistent with a transglutaminase mechanism. gamma-Glutamyl[3H]putrescine could be recovered in 80-90% yield from the proteolytic digest of proteins from the 20-min fertilized cell. Another sign of the in vivo activity of transglutaminase was the isolation of substantial amounts of epsilon-(gamma-glutamyl)lysine from proteins of sea urchin embryo, yielding a frequency value for this cross-link as high as 1 mol/400 000 g of protein in the 32-cell-stage material.