Lurbinectedin is a novel and potent selective inhibitor of active transcription of protein-coding genes, triggering apoptosis of cancerous cells. It has been approved for the treatment of patients with metastatic small-cell lung cancer with disease progression on or after platinum-based chemotherapy. Studies exploring the disposition and metabolism of lurbinectedin were performed in vitro and in vivo (by intravenous administration of lurbinectedin). Low blood cell partitioning for lurbinectedin in rats, nonhuman primates (NHP), and humans was determined as 23.4%, 29.8%, and 9.8%, respectively. Protein binding was very high (>95%) in total plasma (rat, NHP, and human), albumin, and α-1-acid glycoprotein (both human). In vitro, lurbinectedin underwent intense liver microsome-mediated metabolism-in 10 minutes, 80% of the compound is metabolized in human-with CYP3A4 being the isoform involved in that metabolism. Results also showed NHPs being the nonclinical species which, metabolically, most closely resembles humans. Mass balance studies performed in rats (both genders), NHPs (male only), and patients (both genders) demonstrated that the principal route of excretion of 14C-lurbinectedin-related radioactivity was through the feces (88.7% ± 10.1% in patients), with only a minor fraction recovered from the urine (5.6% ± 2.0% in patients). In plasma samples, the majority of lurbinectedin-related radioactivity was attributed to unchanged compound (95% ± 3.1% and 70.2% ± 10.9% in NHPs and humans, respectively). Plasma metabolic profiling demonstrated the major (% compared with unchanged compound) circulating metabolites were N-Desmethyl-lurbinectedin (0.4% ± 0.2% and 10.4% ± 2.2% in NHPs and patients, respectively) and 1',3'-Desmethylene-lurbinectedin (0.9% ± 0.7% and 14.3% ± 10.4% in NHP and patients, respectively). SIGNIFICANCE STATEMENT: Lurbinectedin is a novel and potent selective inhibitor of active transcription of protein-coding genes, triggering apoptosis of cancerous cells, and was recently approved for the treatment of patients with metastatic small-cell lung cancer with disease progression on or after platinum-based chemotherapy. The present study provides a complete set of information on the pharmacokinetics, biotransformation, and elimination of 14C-lurbinectedin and its metabolites, following a single intravenous administration to nonclinical species (rats and nonhuman primates) and patients.