To establish the zebrafish as a model to investigate drug metabolism through sulfation, we had previous cloned, expressed, and purified fourteen distinct zebrafish cytosolic sulfotransferases (SULTs). In the present study, we carried a systematic analysis of the sulfating activities of these fourteen zebrafish SULTs toward a panel of drug compounds. Results showed that four of the fourteen zebrafish SULTs showed no detectable activities toward any of the tested drugs. Among the other ten zebrafish SULTs, three SULT1 enzymes (SULT1 ST1, SULT1 ST2, and SULT1 ST3) displayed considerably stronger activities than the others toward the majority of the drug compounds tested. Specifically, SULT1 ST1, SULT1 ST2, and SULT1 ST3 showed the highest specific activities, at 26.9, 29.3, and 31.5 nmol/min/mg, toward aesculetin, 4-methylembelliferone, and dobutamine, respectively. To further investigate the sulfation of tested drugs by the responsible zebrafish SULT enzymes, the kinetics of the sulfation reactions were analyzed. Kinetic constants determined indicated that the sulfation of these drugs by the SULT enzymes tested is likely to be physiologically relevant. A metabolic labeling experiment using cultured zebrafish liver cells and HepG2 human hepatoma cells was performed. Results showed that zebrafish liver cells displayed a similar pattern of sulfation of the drugs tested as that of HepG2 cells, implying that human and zebrafish liver cells may share considerable similarities with regard to their constituent drug-sulfating SULT enzymes.