A genomic DNA fragment encoding tenecin 3, an antifungal protein was cloned from the genomic DNA library of Tenebrio molitor. The DNA sequence analysis showed that the coding region is divided into two exons by an intron of 49 bp in the middle of the putative leader peptide coding region. Southern blot analysis suggests that the gene is present as a single copy. The transcription initiation site was determined by primer extension analysis and S1 mapping. The TATA box and CCAAT box sequences were found at -30, and -121, respectively, from the transcription initiation site. Tenecin 3 mRNA is abundantly expressed in larvae and adults, while little was detected in RNAs from pupae, suggesting that the expression of the tenecin 3 gene is developmentally regulated.