Steroids are synthesized mainly from the adrenal glands catalyzed by steroidogenic enzymes; the expression of these enzymes is controlled by transcription factor steroidogenic factor-1 (SF-1; NR5A1). To understand the physiological effect of genetic changes on steroid secretion, we used Cre-LoxP and gene targeting technology to mutate the binding sequence for SF-1 (SF-1 response element) on the promoter of the mouse Cyp11a1 gene, which encodes a critical enzyme for steroid biosynthesis. The resulting Cyp11a1 L/L mice expressed about 7-fold less cytochrome P450 side-chain cleavage enzyme (CYP11A1) in the adrenal and testis but expressed normal amounts of CYP11A1 in the placenta and ovary. This tissue-specific reduction of gene expression did not affect basal steroid secretion but attenuated the circadian rhythm of glucocorticoid secretion. These mice also failed to induce glucocorticoid secretion in response to stress, leading to retention of CD4+CD8+ double-positive thymocytes. Unlike complete Cyp11a1 disruption, which causes neonatal death, promoter mutation did not decrease life span and caused no defect in reproduction. Thus, CYP11A1 appears in normal mice to be expressed above the minimal required level, providing a large capacity for use in response to stress. Mutation of the SF-1 response element of Cyp11a1 results in reduced stress response due to decreased adrenal CYP11A1 expression and insufficient stress-induced glucocorticoids secretion.