Menkes fibroblasts contain a significantly greater amount of cysteinerich 10,000 dalton copperbinding protein(s) (metallothionein) than normal cells. Mutant fibroblasts incorporated 30 to 40% more tritiated amino acids into 10,000 dalton protein(s) than normal cells. The protein(s) was deficient in aromatic amino acids. The amount of35S-cysteine incorporated by the same protein(s) in Menkes fibroblasts was twice that of normal fibroblasts. Comparison of the35S:3H isotopic ratios of chromatographic fractions of both normal and Menkes cell lysates showed that only the proteins eluted in the 10,000 dalton peak were enriched in35S-cysteine, and this ratio was always greater in Menkes than in normal cells. The molecular weight35S-cysteine- and3H-amino acidlabeled peaks coincided with the64Cu peak in both cell strains. The copperlabeled peak was always greater in Menkes than in normal cells. No difference in the64Cu:35S isotopic ratio in the 10,000 dalton peak was observed between normal and Menkes fibroblast strains. This finding shows the direct relationship between the amount of cysteinerich 10,000 dalton protein(s) and the amount of64Cu bound by this protein(s) in both Menkes and normal fibroblasts. DEAE cellulose ionexchange chromatography resulted in a further two fold enrichment of the 10,000 dalton, sulfur rich proteins that were eluted from the Sephadex G-75 column. Most of the labeled proteins from both normal and Menkes fibroblasts were eluted from the ion-exchange column in a single peak at a chloride concentration of approximately 30 mM. Polyacrylamide disc gel electrophoresis of pooled fractions of the dalton proteins eluted from the G-75 column and the DEAE-cellulose ion-exchange column showed no consistent differences in the staining pattern between normal and mutant fibroblast strains. When the acrylamide gels were sliced and subsequently counted for radioactive content, no band showed a further increase in the35S:3H isotopic ratio when compared to the electrophoresed samples that were eluted from the Sephadex G-75 or the ion-exchange columns. Also, no significant increase in the amount of radioactivity associated with a specific protein band could be demonstrated between the Menkes and the normal fibroblast strains. Speculation The increased accumulation and the reduced efflux of copper observed in cultured Menkes fibroblasts is associated with a greater amount of copper metallothionein(s) present in the mutant than in the normal cells. The abnormally high copper concentrations observed in several extrahepatic tissues of patients with Menkes disease may also relate to the presence in these tissues of increased levels of copper metallothionein(s).