Dp87 gene in Dictyostelium is a novel prespore-specific gene, whose expression is first observed when the aggregation stream is formed, the earliest among prespore-specific genes so far isolated. By 5'-sequential deletion analyses, we had previously indicated that the region between -447 and -356 is important for transcription. Here we show by detailed analyses that the regulatory mechanism of the gene is more complex in that multiple positive and negative regulatory regions including the previously identified region act cooperatively. In addition, we show that the region including the putative TATA box and the transcriptional start site is required for proper negative regulation of the gene.