Menkes fibroblasts contain a significantly greater amount of cysteine-rich 10,000 dalton copper-binding 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 of35-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 10,000 molecular weight35S-cysteine- and3H-amino acid-labeled peaks coincided with the84Cu peak in both cell strains. The copper-labeled peak was always greater in Menkes than in normal cells. No difference in the84Cu: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 cysteine-rich 10,000 dalton protein(s) and the amount of84Cu bound by this protein(s) in both Menkes and normal fibroblasts. DEAE-cellulose ion-exchange 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. Poly-acrylamide disc gel electrophoresis of pooled fractions of the 10,000 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.