1. Four non-acidic primary metabolites of N-(5-pyrrolidinopent-3-ynyl)succinimide (BL 14) were identified and quantified using g.l.c. and mass spectrometry. The metabolites are alpha-hydroxy-N-(5-pyrrolidinopent-3-ynyl)succinimide (A), N-(5-(2-oxopyrrolidino)-pent-3-ynyl)succinimide (B), N-(2-hydroxy-5-pyrrolidinopent-3-ynyl)succinimide (C) and N-(5-pyrrolidinopent-3-ynyl)succinimide N-oxide (E), the latter analysed after reduction to the parent amine. 2. In rat liver preparations, all metabolites are formed by microsomal, NADPH-dependent enzyme systems, but with different characteristics. The response to inhibitors such as CO and SKF 525A indicates participation of cytochrome P-450 enzymes in the formation of all metabolites. Phenobarbital pretreatment markedly enhances propynylic hydroxylation (C) but has little or no effect on the other metabolic pathways. Succinimide hydroxylation (A) exhibits a pH optimum at 7.0, while the formation of metabolism B and C increases at pH values between 6.4 and 7.7. 3. Kinetic studies on the formation of metabolites A-C revealed differences in the Michaelis constant, while the Vmax values were similar. Succinimide hydroxylation (A) is most efficient with a Km of 3.7 X 10(-5) M, compared with a Km of 1.7 X 10(-3) M for propynylic hydroxylation (C). 4. The formation of metabolites B and E conforms to the corresponding mechanisms for lactam and N-oxide formation for other xenobiotics. The formation of metabolites A and C represents two extremities, reflected in their different responses to phenobarbital pretreatment, pH changes and in their different Km values. Although little can be discerned about the mechanisms from the literature, the enzymes catalysing both reactions appear to be cytochromes.