Serpins are a unique class of serine protease inhibitors that are becoming increasingly recognized as important regulators of insect defense mechanisms and developmental processes. Previously, we identified three Mamestra configurata serpins that were similar in structure to those encoded by the Manduca sexta Serpin-1 gene. To gain insight into the evolution and function of serpins in lepidopterans, we developed a bacterial artificial chromosome library and sequenced the entire M. configurata gene. The Serpin-1 gene was 28 kbp and had the capacity to encode nine serpin isoforms via alternate splicing of exons encoding variant reactive center loops onto a common scaffold. The relative abundance of each isoform was estimated by expressed sequence tag analysis and their expression patterns examined in various developmental stages and larval tissues. The organization of the M. configurata Serpin-1 gene was very similar to that of M. sexta Serpin-1; however, only the Ms Serpin-1Z (1 of 12) and the Mc Serpin-1a isoforms exhibited a high degree of similarity. Orthologs similar to this variant were also found in other lepidopterans, namely Bombyx mori and Plutella xylostella, suggesting that they are involved in a conserved biochemical process and likely represent the ancestral serpin variant. Expansion of the exon family encoding the Serpin-1 reactive centre loop region appears to be a product of recent duplication events that has given rise to different serpin repertoires in related insect taxa.