Cells demonstrating the multidrug resistance phenotype because of overexpression of P-glycoprotein, a drug efflux pump, are resistant to the cytotoxic effects of most natural product drugs. To determine if P-glycoprotein confers resistance to the syctophycins, a family of natural cytotoxic macrolides recently isolated from cyanobacteria of the family Syctone-mataceae, we have characterized the effects of these compounds on drug-sensitive (SKOV3) and drug-resistant (SKVLB1) human ovarian carcinoma cells. While SKVLB1 cells demonstrated > 150- and 10,000-fold decreases in sensitivity to Adriamycin and vinblastine, respectively, they were equally sensitive as SKOV3 cells to the antiproliferative effects of tolytoxin and certain related scytophycins. The SKVKB1 cells were 4- to 11-fold resistant to other scytophycins and were 14-fold resistant to cytochalasin B. Microfilaments in SKOV3 and SKVLB1 cells were depolymerized by similar concentrations of tolytoxin, while cytochalasin B was less potent toward SKVLB1 cells than SKOV3 cells. Both tolytoxin and cytochalasin B enhanced the cytotoxicity of vinblastine toward SKVLB1 cells; however, neither compound affected the sensitivity to Adriamycin or cisplatin. Verapamil markedly increased the accumulation of [3H]vinblastine by SKLVB1 cells, while cytochalasin B caused only modest increase, and tolytoxin had no effect on [3H]vinblastine accumulation. These results suggest that some of the scytophycins, including tolytoxin, are not subject to P-glycoprotein-mediated efflux from cells exhibiting multidrug resistance due to overexpression of this transport protein. These compounds may therefore be useful for killing drug-resistant tumor cells.