This report describes the relationship between the pharmacokinetics, antitumour activity and toxicity of chlorambucil (CHL), phenylacetic acid mustard (PAAM) and beta, beta-difluorochlorambucil (beta-F2CHL) in mice. Pharmacokinetics were studied by HPLC, antitumour activity by a regrowth delay assay using the KHT murine sarcoma and toxicity by acute LD50. For both antitumour activity and acute toxicity the order of potency was: PAAM greater than CHL greater than beta-F2CHL. CHL and PAAM exhibited identical therapeutic indices, whereas that for beta-F2CHL was somewhat improved. CHL is metabolized by mitochondrial beta-oxidation to the 3,4-dehydro derivative (DeHCHL) and PAAM, and the latter is further metabolized to its monodechloroethylated derivative DeC-PAAM, presumably by hepatic microsomal enzymes. Administered PAAM gave only one metabolite, DeC-PAAM. Unexpectedly, despite beta, beta-disubstitution, beta-F2CHL was also beta-oxidized to give DeHCHL and PAAM, but at reduced rates. Further, metabolic switching was demonstrated with the appearance in large amount of 2 new, unidentified metabolites, which may be dechlorethylation products. The pharmacokinetics of administered CHL, PAAM and beta-F2CHL differ in that the plasma clearance was fastest for CHL, slowest for PAAM and intermediate for beta-F2CHL. For the metabolites, CHL produced peak plasma concentrations of DeHCHL and PAAM, respectively, 7-fold and 2-fold greater than those produced by beta-F2CHL. However, despite these differences, exposures to total bifunctional nitrogen mustards were similar following administration of the 3 drugs and therefore cannot account for their differential activity. In contrast, there was a good correlation between potency and PAAM exposure, which is highest after treatment with PAAM, intermediate after CHL and lowest after beta-F2CHL. In plasma, 3.2% of PAAM is present as nonprotein-bound free drug, compared to 1.3% for DeHCHL, 0.9% for CHL and 0.45% for beta-F2CHL. We propose the amount of free bifunctional nitrogen mustard, itself partly dependent on the extent of metabolism, to be of major importance for the in vivo potency of CHL analogues.