Steroid 21-hydroxylase is localized in microsomes of endoplasmic reticulum membranes within adrenal cortex. It is one of three microsomal steroidogenic cytochrome P450 enzymes, the others being steroid 17-hydroxylase and aromatase.
Unlike other enzymes of the cytochrome P450 superfamily of enzymes that are expressed in multiple tissues, with most abundant expression in the liver, in adult humans steroid 21-hydroxylase, along with steroid 11β-hydroxylase and aldosterone synthase, is almost exclusively expressed in the adrenal gland.
 the main subcellular location for the encoded protein in human cells is not known, and is pending cell analysis.