The Drosophila suppressor of position-effect variegation Su(var)3-9 encodes a heterochromatin-associated protein that is evolutionarily conserved. In contrast to its yeast and mammalian orthologs, the Drosophila Su(var)3-9 gene is fused with the locus encoding the gamma subunit of translation initiation factor eIF2. Synthesis of the two unrelated proteins is resolved by alternative splicing. A similar dicistronic Su(var)3-9/eIF-2gamma transcription unit was found in Clytus arietis, Leptinotarsa decemlineata, and Scoliopterix libatrix, representing two different orders of holometabolic insects (Coleoptera and Lepidoptera). In all these species the N terminus of the eIF-2gamma, which is encoded by the first two exons, is fused to SU(VAR)3-9. In contrast to Drosophila melanogaster, RT-PCR analysis in the two coleopteran and the lepidopteran species demonstrated the usage of a nonconserved splice donor site located within the 3' end of the SU(VAR)3-9 ORF, resulting in removal of the Su(var)3-9-specific stop codon from the mRNA and complete in-frame fusion of the SU(VAR)3-9 and eIF-2gamma ORFs. In the centipede Lithobius forficatus eIF-2gamma and Su(var)3-9 are unconnected. Conservation of the dicistronic Su(var)3-9/eIF-2gamma transcription unit in the studied insects indicates its origin before radiation of holometabolic insects and represents a useful tool for molecular phylogenetic analysis in arthropods.