Three members of the human metallothionein-I gene family have been cloned and characterized. Two of the genes encode closely related but distinct metallothionein-I subtypes. Both of these genes are functional as shown by their transcription in cultured hepatoblastoma cells and by their ability to render transfected cells resistant to cadmium toxicity. The cotranscription of these nonallelic genes shows that the previously observed microheterogeneity of metallothionein-I protein preparations is due to the expression of distinct gene products. The third clone is incapable of encoding a typical metallothionein due to an early termination codon and two nonconservative amino acid replacements. This nonfunctional pseudogene retains introns. Evolutionary comparisons reveal conserved DNA sequences in both the coding and regulatory regions of these genes.