We investigated the immunohistochemical localization of aromatase cytochrome P-450 (P-450arom) using a specific polyclonal antiserum (PAb R-8-2). We compared catalytic activity, as detected by the tritiated water assay, and tissue levels of P-450arom, as detected by the specific enzyme-linked immunosorbent assay, in placental samples from early pregnancy to term. Immunostaining and subsequent detection by light and electron microscopy demonstrated that P-450arom is localized in the microvilli and endoplasmic reticulum of the syncytiotrophoblasts of the chorionic villi, but is not present in the mitochondria, nuclei, or cytotrophoblasts at any time during gestation. The P-450arom concentration and aromatase activity were greater in the microsomal fraction than in the mitochondrial fraction or total homogenate at each gestational stage and increased linearly as pregnancy progressed. However, the specific activity of P-450arom was comparable among subcellular fractions in each gestational period. These results suggest that the nature and localization of P-450arom are unchangeable, and the P-450arom concentration increases during pregnancy. It appears that the aromatase detected in the mitochondrial fraction is a contamination of microsomal aromatase, and the increase in aromatase activity can be attributed to an increase in the number of P-450arom molecules rather than an increase in the catalytic ability of each molecule.