The primary structure of metallothioneins (MT) of a mollusc, the oyster Crassostrea virginica, was determined by molecular cloning and mass spectrometry of purified proteins. The cloning strategy included PCR amplification of the responsible cDNAs from total cDNA using completely degenerate oligonucleotides (derived from the N-terminal amino acid sequence) and oligo(dT)20 as primers. Primer extension off mRNA was used as an independent determination of the nucleotide sequence represented by the degenerate PCR primers. The deduced amino acid sequence was consistent with characteristics of class I MT. Twenty-one cysteine residues, were arranged in nine Cys-X-Cys motifs, five as Cys-Lys-Cys. A single Cys-X-X-Cys motif was also observed. Two MTs that differ only in the presence or absence of an N-acetyl group exist in this organism. Masses of tryptic peptides of purified MTs corresponded with those of peptides predicted from tryptic cleavages of the deduced amino acid sequence. Allowing for known N-terminal modifications, 96% of the deduced sequence was confirmed by mass spectrometry. Comparison (FASTA algorithm) of the primary structure of the oyster MTs with those of other species indicated a higher similarity with vertebrate MTs than with those of other invertebrates.