To characterize the transcription regulation of the POU-M1/SGF-3 gene, we have cloned a genomic DNA fragment encompassing the whole coding region and its flanking sequences. This gene does not contain any intron. The 5'-flanking region of the gene contains several interesting motifs, such as homeodomain-binding motifs, sequences resembling the transcriptional factor Sp1-binding site, and TGTTT motifs, but lacks some of the typical transcriptional regulatory sequences, such as TATA and CCAAT boxes. Transcriptional analysis of a series of deletion mutants of the gene in the nuclear extracts prepared from the middle silk gland of 2-day-old fifth instar larvae revealed the presence of multiple cis-regulatory elements located both upstream and downstream of the initiation site. One of these elements, the homeodomain-binding element, was identified to mediate negative regulation. By mobility shift assay using the POU-M1 specific antibodies, we found that this negative element interacts with the POU-M1/SGF-3. Transcription analysis in vitro using templates mutagenized in the PB region and one of the POU-M1 antibodies indicated that the PB region is an autoregulatory element responsible for SGF-3-dependent transcriptional repression.