The highly cytotoxic, sponge-derived, antimitotic macrolide polyether spongistatin 1 has been previously shown to inhibit microtubule assembly, the binding of vinblastine and GTP to tubulin, and displacement of GDP bound in the exchangeable site of tubulin. We have now examined in detail inhibition by spongistatin 1 of both [3H]vinblastine and [3H]dolastatin 10 binding to tubulin. We found spongistatin 1 to be a noncompetitive inhibitor of the binding of both radiolabeled drugs to tubulin, in contrast to competitive patterns obtained with vincristine versus [3H]vinblastine and with a chiral isomer of dolastatin 10 versus [3H]dolastatin 10. Since dolastatin 10 is itself a noncompetitive inhibitor of vinca alkaloid binding to tubulin, this implies at least three distinct binding sites for the structurally complex and diverse natural products that interfere with each others binding to tubulin and with nucleotide exchange. Spongistatin 1, in contrast to both vinca alkaloids and peptide antimitotic agents like dolastatin 10, does not induce formation of a GTP-independent, morphologically distinctive polymer ("aggregate"). We also examined eight compounds closely related structurally to spongistatin 1 (spongistatins 2-9). The most distinctive in their properties were spongistatins 6 and 8. These two compounds, despite activity comparable to spongistatin 1 as inhibitors of tubulin polymerization and [3H]vinblastine binding, had much reduced activity as inhibitors of nucleotide exchange and [3H]dolastatin 10 binding. Spongistatins 1 and 6 were compared for effects on dolastatin 10-induced aggregate formation in conjunction with effects on [3H]dolastatin 10 binding. Spongistatin 6 was about 4-fold less active than spongistatin 1 as an inhibitor of aggregation and over 20-fold less active as an inhibitor of dolastatin 10 binding.