Caspase-3 is the major effector in apoptosis triggered by various stimuli. Previous studies demonstrated a significant increase in transcriptional activity of the caspase-3 gene during neuronal apoptosis. Recent findings suggest that differential expression of the caspase-3 gene may underlie the regulation of apoptotic susceptibility during brain development and after acute injury to the mature brain. We identified and cloned the rat caspase-3 gene promoter, determined its structure, and examined its regulation during a course of apoptosis in PC12 cells. Results demonstrate that this promoter lacks a TATA-box and contains a cluster of Sp1 elements and multiple transcription start sites. The first identified transcription start site is located 87-bp upstream from the first splicing site. A role of Sp1 elements in the regulation of caspase-3 promoter activity is demonstrated by the inhibition of Sp1 binding using mithramycin A. Results of deletion analysis show that an Ets-1-like element located between nucleotides -1646 and -1632 relative to the most extended transcription start site is necessary to achieve sustained transcriptional activity. Homology analysis revealed that the 5'-flanking region of the human caspase-3 gene exhibits significant similarity to a regulatory region of the rat gene.