Nebulin family genes are believed to have diverged from a single gene during the evolution of vertebrates. We determined the structure of the amphioxus nebulin gene and showed that in addition to the features of the human nebulin gene, this gene had a LIM domain, secondary super repeats and a giant exon with 98 nebulin repeats containing unique sequences. A transcript of this gene amplified by reverse transcriptase-polymerase chain reaction had a LIM domain, three nebulin repeats and an SH3 domain. This transcript was similar to an isoform of human nebulette (Lasp-2). Phylogenetic analysis using the LIM and SH3 domains of the nebulin family proteins showed that amphioxus nebulin is located outside the vertebrate nebulin family group in the phylogenetic tree. These results indicated that the amphioxus nebulin gene had a unified structure among nebulin, nebulette, lasp-1 and N-RAP of vertebrates, and that these nebulin family genes diverged from the amphioxus nebulin gene during the course of vertebrate evolution.