Two A-ring substituted derivatives of camptothecin, the 9-amino and 10,11-methylene-dioxy derivatives, were shown to be potent inhibitors of topoisomerase I with demonstrated in vivo efficacy against human tumor xenografts selected from the NCI in vitro screen. Of these, 9-aminocamptothecin was recently selected by DTP for clinical evaluation. Our recent studies have been concerned with a comparison of the murine pharmacokinetics of these substituted derivatives to those of camptothecin lactone and the sodium salt of the opened lactone ring of camptothecin. Employing a novel HPLC assay developed for these studies, the murine pharmacokinetics of 9-aminocamptothecin were found to be markedly dissimilar from that of camptothecin or its 10,11-methylenedioxy derivative. The far smaller steady state volume of distribution of the 9-amino compound suggests that its tissue binding relative to plasma protein binding is far less than that of the other compounds and results in relatively higher total body plasma clearance. The different structural modifications of the A-ring of camptothecin markedly influenced the pharmacokinetics. Continued studies with camptothecin derivatives will serve to aid the development of this important therapeutic class.