Using the technique of direct sample insertion selected ion monitoring chemical ionization mass spectroscopy, BCNU levels were measured in vivo in patients and in vitro in serum, sera ultrafiltrates, and buffered Ringer's solution. The disappearance of BCNU in vitro was found to be first-order with a half-time of 11.6 minutes (+/- 0.5 SD) in volunteers and 15.6 minutes (+/- 2.3 SD) in patients. The disappearance from serum was catalyzed by a macromolecular component of the serum and was slowed by serum lipids. Analysis of the pharmacokinetics of BCNU in 20 patients using a two-compartment open model demonstrated a volume of distribution of 3.25 liters/kg (+/- 1.69 SD), a clearance of 56 ml/minute/kg (+/- 56 SD), and a transfer constant from the central compartment to the outside (K10) OF 0.0324 MINUTE-1 (+/- 41% SD), which was close to the decomposition rate observed for BCNU in serum in vitro. The pharmacokinetics of BCNU in patients may be affected by the percent of body fat and the lipid content of the serum.