A simple and rapid analytical method was established for detection of anabolic agents, zeranol and alpha-trenbolone and beta-trenbolone residues in cattle muscle and liver. This method consisted of aqueous acetonitrile extraction and successive solid-phase extraction with a tandem connected octadecyl (C18)-aminopropyl (NH2) cartridge, followed by on-line clean-up using multidimensional HPLC with EC and UV detection. On-line clean-up was performed by a three-column switching technique, the first column being a Diol size exclusion column (SEC), the second, an internal surface reversed phase trapping column (TC) and the third, a C18 analytical column (AC). Sample solution from solid-phase extraction was injected into the SEC. The anabolic agents eluted from the SEC were introduced into a TC, and the anabolic agents trapped in the TC were rapidly loaded onto an analytical column after changing the eluent. Zeranol in the eluate from the analytical column was detected at 700 mV ECD and alpha- and beta-trenbolone were detected at UVD 350 nm. SEC and TC were washed and conditioned to the initial state just after the loading of the desired fraction on the AC. Recoveries from muscle fortified at a level of 0.002 mg/L (maximal residue limits, MRLs) averaged 86.1% for zeranol and 77.0% for beta-trenbolone, and recoveries from liver at a level of 0.010 mg/kg (MRLs) averaged 74.3% for zeranol and 73.1% for alpha-trenbolone. The quantitation limits for all of them were 0.001 mg/kg in muscle and 0.002 mg/kg in liver.