We have used a transient expression assay employing Drosophila S2 cells to study the transcriptional repression activity of a 27 amino acid residue-long repression domain FS1 which was generated by a frame-shift in a pair-rule gene, even-skipped of Drosophila melanogaster. In an attempt to define a minimal requirement for the repression activity, we constructed a series of truncation mutant forms of the FS1, fused to a heterologous GAL4 DNA-binding domain, and measured their activities. All of the mutant forms, including the GAL4-FS1 (5-23) which retains the smallest number (19) of amino acid residues of FS1, were found to repress an initiator, a minimal TATA-lacking promoter, in a GAL4-binding-site-dependent manner. These findings suggest that a 19 amino acid residue-long region, rich in proline and leucine residues, is a transcriptional repression domain and may interact with the general transcription machinery.