An embryonic enhancer determines the temporal activation of a sea urchin late H1 gene. Normal development requires that individual genes be expressed in their correct temporal patterns, but the mechanisms regulating this process during early embryogenesis are poorly understood. We have studied the early and late sea urchin histone genes during embryogenesis to address the molecular mechanisms controlling temporal gene expression. By measuring the changes in expression of cloned H1-beta DNA constructs after microinjection into fertilized one-cell zygotes, we demonstrated that a highly conserved 30-base-pair segment of DNA between positions -288 and -317 (USE IV) is responsible for the transcriptional activation of this late histone gene at the late blastula stage. In this report, we demonstrate that an oligonucleotide corresponding to USE IV acts as an embryonic enhancer element capable of activating the simian virus 40 early promoter in a stage-specific manner. Using an in vivo competition assay and in vitro DNase I footprinting and mobility shift assays, we also identified a protein(s) that interacts with this enhancer. Results of the competition assay suggested that this factor acts to stimulate transcription of the H1-beta gene. The factor was found to be stored in mature eggs as well as in all embryonic stages examined. The mobility of the factor found in eggs, however, differed from that of the embryonic form, which suggested that posttranslational modification occurs after fertilization.