The dimeric pyrrolobenzodiazepine SJG-136 (NSC 694501) has potent in vitro cytotoxicity and in vivo antitumor activity. SJG-136 binds in the minor groove of DNA and produces G–G interstrand cross-links via reactive N 10 –C 11 /N 10′ –C ll′ imine/carbinolamine moieties. We have developed a sensitive, specific liquid chromatography tandem mass spectrometry (LC/MS/MS) method for the quantitative determination of SJG-136 in plasma. SJG-136 was isolated by solid phase extraction through a C8 column, reverse-phase HPLC separation was accomplished on a C18 column with isocratic elution and MS/MS detection, monitoring the m/z 557–m/z 476 transition after electrospray ionization. The linear range and lower limit of quantitation from plasma standard curves were 2.8–1800nM, and 5nM, respectively. SJG-136 plasma protein binding was species-dependent. Values of the unbound fraction in human, rat and mouse were 25%, 16.2% and <1%, respectively. Protein binding was saturable in dog plasma where the unbound fraction increased from 10.8% to 22.3% over a 22–720nM concentration range. SJG-136 pharmacokinetics after a single intravenous dose were best fit to a two-compartment open model with elimination half-life and plasma clearance values of 97min and 6.1mL/min/kg, respectively. SJG-136 did not accumulate in plasma following intravenous administration of 1.0μg/kg doses for five consecutive days.