Pregnenolone is synthesized from cholesterol. This conversion involves hydroxylation at the side-chain at C20 and C22 positions, with cleavage of the side-chain. The enzyme performing this task is cytochrome P450scc, located in the mitochondria, and controlled by anterior pituitary tropic hormones, such as adrenocorticotropic hormone, follicle-stimulating hormone and luteinizing hormone.

Pregnenolone can be converted to 17-hydroxy-pregnenolone by the enzyme 17_-hydroxylase (CYP17A1). Using this pathway, termed delta-5 pathway, the next step is conversion to dehydroepiandrosterone (DHEA) using a desmolase. DHEA is the precursor of androstenedione.

Pregnenolone and its sulfate, like DHEA and its sulfate and progesterone, belong to the group of neuro-steroids that are found in high concentrations in certain areas of the brain, and are synthesized there. Since pregnenolone is a precursor to DHEA, its balance in the body helps to support memory and cognition, joint health, energy, fatigue reduction, cholesterol balance and wellness and mood.