Inconsistencies have been reported on the effect of sex on aldosterone levels leading to clinical confusion. The reasons for these inconsistencies, are uncertain but include: estrogen and/or its receptor modulating target gene responses to mineralocorticoid receptor activation and aldosterone secretagogues’ levels. This study’s goal was to determine whether aldosterone’s biosynthesis also differed by sex. Two approaches were used. First, plasma renin activity (PRA) and aldosterone were measured in rats. Both were significantly higher in males. Secondly, using rat zona glomerulosa (ZG) cells, we assessed three ex-vivo areas:1) activity/levels of early steps in aldosterone’s biosynthesis (StAR and CYP11A1); 2) activity/levels of a late step (CYP11B2); and 3) the status of the MR mediated, ultrashort-feedback-loop. Females had higher expression of CYP11A1 and StAR; and increased CYP11A1 activity (increased pregnenolone/ corticosterone levels) but did not differ in CYP11B2 expression or activity (aldosterone/ levels). Activating the ZG’s MR (thereby activating the ultrashort-feedback-loop) reduced CYP11B2’s activity similarly in both sexes. Ex-vivo, these molecular effects were accompanied, in females, by lower aldosterone basally but higher aldosterone with angiotensin II stimulation. In conclusion, we documented that not only was there a sex-mediated difference in the activity of aldosterone’s biosynthesis, but also these differences at the molecular level, help explain the variable reports on aldosterone’s circulating levels. Basally, both in-vivo and ex-vivo, males had higher aldosterone levels, likely secondary to higher aldosterone secretagogue levels. However, in response to acute stimulation, aldosterone levels are higher in females because of the greater levels and/or activity of their StAR/CYP11A1.