AbstractTwo aromatase genes have been discovered in the brain and ovary of some teleosts. However, data on native aromatase enzyme kinetics and thus actual catalytic activity are scarce in fish, impeding comparison of aromatase activity (AA) from different organs within and between species. In the present study, the tritiated water assay was optimized and validated to measure AA in the sea bass using 1β‐[3H]‐androstenedione as a substrate in crude homogenates and microsomes. Optimized assay variables included pH, temperature, buffer strength, incubation time, amount of fresh tissue, substrate, and cofactor concentration. Specificity of the assay was verified by using known inhibitors, inappropriate substrates, and heat‐inactivation. Subcellular fractionation revealed ten‐fold more activity in the microsomal over the cytosolic fraction. The assay was also validated by comparing results from the direct product isolation method. The validated assay described allows measurement of AA to levels as low as < 10 fmol/mg protein/hr. Sex differentiation is temperature‐dependent in the sea bass. It was found that in the physiological range of temperatures where the sea bass can live, 10–30°C, AA is highly dependent on temperature in a linear fashion (brain: r2 = 0.92; P<0.001; ovary: r2 = 0.94; P<0.001). When AA levels from brain and ovarian homogenates obtained from the same fish during the spawning season were compared, the respective Michaelis‐Menten constant (Km) values were 7.3 nM vs. 4.6 nM, with no significant differences detected between the two tissues. Thus, sea bass aromatase has a very high affinity for androstenedione, similar to what has been found in goldfish, but much higher than other piscine or mammalian aromatases (30–435 nM). In contrast, the brain maximum reaction rate (Vmax 7.8 pmol/mg protein/hr) was four‐fold higher (P<0.001) than the ovarian Vmax (2.1 pmol/mg protein/hr). Consistent results were found using purified microsomes. Although this is the first time that the kinetic parameters are reported for a native piscine aromatase in two different tissues within the same fish, it remains to be determined whether this is a reflection of two distinct isoforms in this particular species. J. Exp. Zool. 293:500–510, 2002. © 2002 Wiley‐Liss, Inc.