The rate of [35S]cystine incorporation into hepatic zinc-thionein (a metallothionein) was stimulated, with a maximum of 5-6h, after parenteral administration of 2mg of Zn2+ containing 65Zn. The binding of 65Zn to zinc-thionein was measurable by 2-1/2h and reached a plateau by 18h after the injection. A net increase in the hepatic 65Zn content was observed subsequent to the decrease in the rate of zinc-thionein synthesis. The incorporation of both 65Zn and [35S]cystine into zinc-thionein was inhibited by prior administration of either actinomycin D or cordycepin. A second injection of Zn2+, 20h after the initial injection, yielded a 4.9-fold greater increase in zinc-thionein synthesis compared with that after only one injection; however, this synthesis was also inhibitable by actinomycin D. These data support the concept that hepatic zinc-thionein synthesis responds quickly to changes in Zn2+ status and that Zn2+ is bound subsequent to synthesis of nascent thionein chains. The mechanism of control of zinc-thionein synthesis by Zn2+ appears to involve changes in the amounts of a short-lived, poly(A)-containing RNA whose translation can be derepressed by additional exposure to Zn2+.