Metallothionein (MT) fractions isolated from human adult liver tissue are readily separated by anion-exchange chromatography in two isoforms, MT-1 and MT-2, which differ from each other in the nature of the amino acid residue at position 11. In fetal liver tissue, the presence of a third charge-separable MT isoform has been previously reported. We determined its partial amino acid sequence and the sequence of a cDNA clone encoding this MT form. This confirmed the existence of another human MT isoform, hereafter named MT-0, which is characterized by the presence of a negatively charged amino acid at position 8, and by a Glu23 to Lys substitution in a strictly conserved region of the protein. Taking into account these substitutions, we are able to classify human MT isoforms into three instead of two charge-separable groups, based on the nature of three amino acid residues. The unique presence of Glu8 in MT-0 enabled us to develop an MT-0-specific amplification by the polymerase chain reaction, which revealed the presence of MT-0 mRNA in adult liver RNA samples, in spite of the total absence of this isoform at the protein level. This suggests the involvement of post-transcriptional regulatory mechanisms in the expression of this fetal MT form.