The genomic structure of the Cypridina luciferase gene in Vargula hilgendorfii (formerly Cypridina hilgendorfii) was determined with three λ phage clones (λ34, λ45, and λ61). The luciferase genes in clones λ34 and λ61 consisted of 13 exons and 12 introns, and clone λ45 only contained exons 1-5. The splicing sites of the luciferase genes in λ34 and λ61 were conserved completely with the consensus sequence. The translated luciferases had 555 amino acid residues, which were over 98.6% identical to those of cDNA clones as previously reported. In contrast, each intron in clones λ34, λ45, and λ61 varied significantly in length. To explain the variation of intron length among the three V. hilgendorfii luciferase genes, genomic DNA was isolated from a single V. hilgendorfii specimen, and the regions from exon 1-3 of the luciferase gene were amplified by polymerase chain reaction (PCR). PCR products with various lengths were detected and were confirmed as the luciferase gene fragments by Southern blot analysis. Furthermore, DNA sequence analysis indicated that at least seven luciferase gene groups might be present in the genome of a single specimen. Thus, multiple Cypridina luciferase genes exist in the genome of a single V. hilgendorfii specimen.