S16020-2 is a new olivacine derivative which has recently shown a marked antitumor activity in various experimental models. This study was undertaken in order to measure the inhibition of the proliferation of various sensitive and resistant tumor cell lines, by S16020-2, and to obtain information concerning its mechanism of action. For a continuous exposure, S16020-2 was as cytotoxic as adriamycin (ADR) (mean IC50 of about 28 nM) and on average, 46 fold more potent than elliptinium acetate (ELP), against a panel of 20 non-multidrug resistant cell lines. With a short exposure (1 hour) followed by a post-incubation of 95 hours in drug-free medium, S16020-2 was 5 and 6 fold more cytotoxic than ADR for human lung A549 and murine melanoma B16 cells, respectively. Furthermore, S16020-2 inhibited more actively the formation of colonies issued from proliferating cells, compared to colonies issued from quiescent A549 cells. Because quiescent cells demonstrated a 3 fold lower level of topoisomerase II alpha (topo II) than proliferating cells, these results suggest that this enzyme could be a potential target for S16020-2. In addition, as demonstrated by flow cytometric studies, S16020-2 intercalated into DNA and induced a cell cycle arrest in G2. Cell lines displaying the multidrug resistance (MDR) phenotype, P388/ADR-1, P388/ADR, P388/VCR-20, KB-A1, DC-3F/AD, S1/tMDR, and Colo320DM, were more sensitive to S16020-2 than to ADR or ELP, as shown by the mean resistance factors, 8, 201, and 23 respectively. In addition, the two cell lines displaying the pure classical MDR phenotype, linked exclusively to the P-glycoprotein (P-gp) overexpression (P388/VCR-20 and S1/tMDR), were as sensitive to S16020-2 as their sensitive parental counterparts, although they were resistant to ADR. S16020-2 is thus one of the most potent olivacine and ellipticine derivative yet characterized. The good cytotoxicity of S16020-2 against cells displaying a P-gp-mediated multidrug resistance, and its antitumor activity in vivo delineate an important chemotherapeutic potential for this drug.