A new and convenient two-step synthesis of 11-substituted 6-amino-11,12-dihydrobenzo[c]phenanthridines and 6-aminobenzo[c]phenanthridines (BPs and BP-Ds) was developed recently in the authors' laboratory. These compounds revealed a high antitumoral activity in in vitro and in vivo test systems. In particular, 11-phenyl-substituted derivatives with two or three methoxy groups showed good activity. It was not clear if the dihydro-derivatives (BPs) were transformed enzymatically into the phenanthridines (BP-Ds), thus acting as prodrugs. The in vitro metabolism of several of these cytostatically active 6-aminobenzo[c]phenanthridines was investigated using human and porcine liver microsomes and a range of expressed human cytochrome P450 enzymes. High-performance liquid chromatography and liquid chromatography-mass spectrometry analysis were used for the quantification and structural identification of the observed metabolites. Aromatic hydroxylation was observed to be the major metabolic pathway in addition to a number of other metabolites. The formation of N-hydroxy- and 6-oxo-derivatives was detected only in very small amounts. BP derivatives are not prodrugs of BP-Ds and no significant differences between human and porcine microsomes were observed, confirming the pig as a good model for metabolism studies of these compounds.