The Japanese pufferfish Fugu rubripes (Fugu) has a small genome of about 400 Mb. Nine different actin genes have been isolated and sequenced from a genomic library constructed from this teleost. The six muscle-type actin genes include two alpha-skeletal actins, three alpha-cardiac actins and an alpha-anomalous (testis type) actin, and the three cytoplasmic actins include two beta-cytoplasmic actins and a beta-cytoplasmic (vascular type) actin. The two skeletal muscle actin genes have identical genomic organization, but differ by five amino acid residues. The three cardiac actin genes code for the same protein but differ in their nucleotide sequences and genomic organization. beta-Cytoplasmic actin1 differs by three amino acids from beta-cytoplasmic actin2. The alpha-anomalous (testis type) and beta-cytoplasmic (vascular type) actins are novel vertebrate actins. The amino acid sequence of alpha-anomalous (testis type) actin is the most divergent of all the known vertebrate actins and transcripts of this gene are abundant in the testis. The beta-cytoplasmic (vascular type) actin gene has eight introns, similar to mammalian smooth muscle actins, and is expressed in vascular tissues such as the gills, kidney and skin. Several known regulatory elements are found in the 5' flanking sequences and the first intron of various Fugu actin genes. The intron patterns of the various Fugu actins seem to be the result of loss of certain introns from a common ancestral gene.