We identified 5' upstream enhancers of two Ci-ZicL genes and characterized one of them in detail. Although the genes are tandemly repeated in the genome, the transcription of each seemed to be individually regulated. The 259-bp 5' flanking sequence contained essential elements for driving a correct spatiotemporal expression. This enhancer can be divided into two distinct modules. The A module was located between nucleotide positions -259 and -205 upstream of the putative transcription start site, and was necessary for activation in A6.2 and A6.4 blastomeres at the 32-cell stage. The BM module lay between nucleotide positions -205 and -89 and was responsible for activation in B6.2 and B6.4 blastomeres at the 32-cell stage and in A-line presumptive notochord, nerve cord, and muscle lineage cells at later stages. Two putative Fox-binding sites, one located within and the other downstream of the BM module, were necessary for the latter activity. Mutation at a potential Ets-binding site, located downstream of the BM module, caused ectopic activation of the reporter gene in a-line presumptive ectoderm cells. This suggests that repression in the a-line blastomeres is necessary for correct transcriptional control of the Ci-ZicL gene.