[1β-³H]16α-Hydroxyandrostenedione (16α-OHA) (715 mCi/mmol) was prepared from commercially available [1β-³H]androstenedione (A) by the microbiological method with Streptomyces roseochromogenes and its structure and purity were determined by chromatographic and reverse isotope dilution methods. When [1β-³H]16α-OHA was incubated with human placental microsomes and reduced nicotinamide adenine dinucleotide phosphate (NADPH), ³H₂O-release into the medium was dependent upon protein concentration and incubation time. An apparent K_m and and V_max of the microsomal aromatase for the [1β-³H]substrate were 650 nM and 34 pmol/min/mg protein, respectively. In this assay, aromatase activity could be determined as low as 0.1 nmol estrogen formation/min/mg protein. 3-Deoxyandrostenedione, a potent competitive inhibitor of the A aromatization, also blocked the 16α-OHA aromatization in a competitive manner with K_i of 15 nM.