The single step chromatographic product isolation method using [4-14C]-androstenedione and the tritiated water method using either [1 beta, 2 beta-3H]- or [1 beta-3H]-androstenedione have been used to determine a suitable method to measure the aromatase activity in rat ovarian 1000 g supernatant and human placental microsomes. The single step product isolation method using [4-14C]A4 reveals the presence of four distinct [4-14C]-labelled products in the rat ovary of which only the synthesis of estradiol is markedly inhibited by CGS 16949A, a well established aromatase inhibitor. In the human placenta, the formation of both [4-14C]-estrone and [4-14C]-estradiol is strongly inhibited by CGS 6949A. Therefore, in the rat ovary spurious results are obtained if accumulative radiolabelled product formation is measured without characterisation of the products. The Vmax in the rat ovary using [1 beta, 2 beta-3H]-A4 as a substrate is 13.7 pmol/h/mg compared to 2.9 pmol/h/mg when [1 beta-3H]-A4 is used. In the human placenta, the Vmax is similar using either [1 beta, 2 betat-3H]-A4 or [1 beta-3H]-A4 (1.21 and 1.27 nmol/h/mg, respectively). Consistent results are obtained for the human placenta using either the single step chromatographic product isolation method or the tritiated water method. However, in the rat ovary the more suitable method of the two used to measure the aromatase activity is the tritiated water method employing [1 beta-3H]-A4 as a substrate. Aromatase activity in the rat ovary during estrus cycle was measured using the tritiated water method employing [1 beta-3H]-A4 as a substrate. A peak of aromatase activity at proestrus was seen which returned rapidly to its basal level at estrus. Plasma estradiol concentrations were in parallel with the aromatase activity.