The metabolism of copper in the yeast Saccharomyces cerevisiae has been studied with respect to the distribution and stability to exchange of newly arrived 64Cu. Cells pre-incubated with 10 microM-Cu2+ accumulated 64Cu into two pools distinguishable by cellular locale and lability to exchange with extracellular cold copper. One pool was non-exchangeable and was localized to protoplasts. Size-exclusion chromatography of a soluble cell (protoplast) extract showed that this 64Cu was associated with up to four species. Two were identified as copper metallothionein and Cu,Zn superoxide dismutase based on comparisons of chromatograms derived from strains in which the genes for these two proteins had been deleted. A third species was identified as copper-glutathione based on chromatographic and biochemical assays. A second pool was exchangeable and was localized to the cell wall. In contrast to its rapid copper-stimulated exchange (t1/2 approximately 1 min), this pool exhibited only slow efflux (10% 64Cu loss per 60 min). Zn2+ did not stimulate the loss of 64Cu from this pool indicating that it was selective for copper. This pool was released into the supernatant upon protoplast formation and was found in the cell wall debris obtained when cells were mechanically disrupted. This 64Cu eluted in the void volume (peak Pv) of the column used to size-fractionate copper-binding species. The metal in Pv was exchangeable in vivo and in vitro. However, the corresponding chromatographic fraction obtained from copper-naive cells when labelled in vitro could bind less than 20% of the 64Cu bound to it in vivo indicating that the deposition of copper in this pool was primarily cell-dependent. In fact, this deposition was shown to be dependent on the cellular reduction of medium sulphate or sulphite to the level of sulphide, or on the addition of sulphide to the 64Cu uptake buffer. 64Cu in the non-exchangeable protoplast pool was not mobilized by cellular sulphide generation, indicating that cellular sulphide generation did not causally lead to the partitioning of 64Cu to the cell wall pool. The data indicate that the appearance of copper sulphide(s) on the cell wall in S. cerevisiae is gratuitous and does not represent a sulphide-based mechanism of copper resistance in this yeast.