Cisatracurium, (1R, 1′R, 2R, 2′R)-2,2-[1,5-pentanediylbis-[oxy(3-oxo-3,1-propanediyl)]]bis[1-[(3,4-dimethoxy-phenyl)-methyl]-1,2,3,4-tetrahydro-6,7-dimethoxy-2-methylisoquinolinium] dibenzenesulphonate (51W89), is an intermediate-acting neuromuscular blocking agent. 51W89 is one of ten isomers contained in Tracrium® (atracurium besylate) and represents' approximately 15 percent of the atracurium mixture. Clinical studies have indicated that 51W89 is more potent and is significantly weaker as a histamine releaser than atracurium. In vitro studies in human plasma have shown that, like atracurium, 51W89 spontaneously degrades at physiological pH by Hoffmann elimination to form laudanosine and the quaternary monoacrylate. Subsequent ester hydrolysis of the monoacrylate generates the monoquaternary alcohol. In rat plasma, 51W89 is also metabolized by non-specific carboxylesterases to the monoquaternary alcohol and the monoquaternary acid, the former being rapidly hydrolysed further to the more stable acid. It has been reported that laudanosine can be further metabolized via N-demethylation to yield tetrahydropapaverine. The rate-limiting step in the degradation of 51W89 in human plasma is Hofmann elimination, whilst in rat plasma, the action of non-specific carboxylesterases is rate limiting. As part of the development of 51W89, the disposition of 14C-51W89 following a single intravenous bolus dose was studied in various animal species and humans. In the present work, we describe the identification of 51W89 metabolites in urine and bite from these studies by high performance liquid chromatography/mass spectrometry using pneumatically-assisted electrospray ionization coupled to an on-line radioactivity monitor. This methodology enabled rapid and sensitive screening of biological samples with minimal sample preparation. Structural confirmation of metabolites was obtained by tandem mass spectrometry. In addition to the expected metabolites, a number of urinary and biliary O-glucuronic acid conjugate of monodesmethyl laudanosine and monodesmethyl tetrahdropapaverine were identified, which cochromatographed with an early eluting ‘unknown’ in the radioprofile. A sulphate conjugate of monodesmethyl laudanosine was also identified in cat bile. The characterization of these metabolites was assisted by the incorporation of on-line radioactivity monitoring during mass spectroscopic analysis, which provided an invaluable means to distinguish drug-related and endogenous material.