The metabolism of N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide (RP73401), a phosphodiesterase IV (PDE IV) inhibitor is extensive (unpublished); however, until recently, studies for this compound did not report 4-amino-3,5-dichloropyridine (ADCP) as a metabolite either in vitro or in vivo. This prompted a reinvestigation into the metabolism of RP73401 in rats and mice using mass spectrometry. The results of the reinvestigation confirmed that 4-amino-3,5-dichloropyridine was formed via the metabolism of RP73401 both in vitro and in vivo. In order to further investigate RP73401 hydrolysis in vivo, a liquid chromatography/mass spectrometry assay was developed and validated for the simultaneous determination of RP73401 and ADCP in rat and mouse plasma. The method used Waters Oasis HLB brand solid phase extraction cartridges to isolate the analytes (RP73401 and ADCP) and internal standard from the plasma. HPLC chromatographic separation was achieved using a Zorbax SB C18 HPLC column and detection was accomplished using positive ion atmospheric pressure chemical ionization tandem mass spectroscopy in multiple reaction monitoring (MRM) mode. The assay was developed and validated over the range of 0.5-100 ng ml(-1) for RP73401 and 5-500 ng ml(-1) for ADCP using 0.050 ml of plasma. The assay proved to be sensitive, accurate, precise and specific for RP73401 and ADCP. Intraday and interday quality control results routinely showed accuracy and precision to be within +/- 20%. This LC/MS/MS method was subsequently employed to investigate the hydrolysis of RP73401 in the rat and mouse, and determine the effects of tri-o-tolyl phosphate (TOTP, a carboxylesterase inhibitor) preadministration on the hydrolysis reaction in the rat.