Trimethylamine-N-oxide (TMAO) is abundant in marine fish. Formaldehyde synthesis by TMAO demethylation during storage markedly deteriorates fish meat. In the present work, we cloned the extremely aspartic acid-rich proteins from skeletal muscle of a commercially important species, walleye pollack, in the course of molecular identification of trimethylamine-N-oxide demethylase (TMAOase). One of the cDNAs, designated as aspolin1, encodes an extremely aspartic acid-rich protein of 228 amino acids which is converted to the TMAOase after processing between Ala42 and Asp43. Mature aspolin1/TMAOase protein contains 179 Asp in 186 total amino acids. The other cDNA, designated as aspolin2, has a common nucleotide sequence with aspolin1 in the 5' part and encodes a protein which has an additional Asp polymer and a C-terminal cysteine-rich region. The amino acid sequence of the C-terminal cysteine-rich region of aspolin2 is highly homologous to the mammalian histidine-rich Ca2+-binding protein. Aspolin1/TMAOase and aspolin2 mRNA was most abundant in the skeletal muscle. A lower level of the mRNA was also detected in kidney, heart, spleen, and brain. Synthetic Asp polymer showed marked TMAOase activity in the presence of Fe2+, whereas a monomer and oligomers did not. Purified TMAOase protein bound to Fe2+ with low affinity, which may be responsible for the catalytic activity. Poly aspartic acid-Fe2+ complex generated after death would be involved in formaldehyde synthesis by the demethylation of TMAO during the storage of fish meat.