Chemical and biological investigations of extracts from the sponge genus Auletta and two collections of Siphonochalina sp. have shown these organisms to be producers of the potent hemiasterlin class of antitumor agents. In addition to the previously known hemiasterlin (1) and hemiasterlin A (2), a new analogue, hemiasterlin C (3), was isolated and identified. The structures of 1 and 2 were assigned based on comparison to literature values, and 3 was identified on the basis of 1H NMR, 13C NMR, COSY, HSQC, and HMBC experiments. The cytotoxic and antitubulin activities of 1-3 were evaluated. In a comparative assay for inhibition of tubulin polymerization, the hemiasterlins were more potent than dolastatin 15 and equipotent with cryptophycin 1, but were somewhat less potent than dolastatin 10.