The metabolism of methylseleninic acid in isolated rat hepatocytes was investigated. Selenium containing metabolites excreted from the cells were detected in the supernatant of the incubation sample by LC-ICP-MS. After pre-treatment of the supernatant by preparative chromatography and pre-concentration by lyophilisation, a major metabolite was identified by molecular mass spectrometry as Se-methylselenocysteine by LC-ESI-MS, MS2 and MS3 and a minor metabolite was identified as selenomethionine by LC-ESI-MS2 and MS3 and LC-ESI-MS2(SRM). This is the first time these metabolites have been identified in hepatocytes. Complementary data from ion trap and triple quadrupole MS instruments provided solid proof of metabolite identities. A time course study showed that S-(methylseleno)cysteine and S-(methylseleno)glutathione were intermediates in the formation of the major metabolite. It is questioned if methylseleninic acid is a relevant model compound for methylated Se-amino acids in vitro.