The microtubule stabilizing agent peloruside A binds to a unique site on the tubulin alpha,beta-heterodimer compared to taxoid site drugs such as paclitaxel (Taxol), docetaxel (Taxotere), epothilone A, and discodermolide. Because the binding sites differ, peloruside A may be able to synergize with these taxoid site drugs when added in combination to cultured cells. Ovarian carcinoma cells (1A9) and myeloid leukemic cells (HL-60) were treated with different concentrations of peloruside A and taxoid site drugs, both compounds given singly and in combination in the nanomolar range, and the antiproliferative activity, G2/M blocking potency, and microtubule stabilizing activity of the treatments assessed. Cell proliferation was monitored using the MTT cell proliferation assay, cell cycle block was determined by flow cytometry, and stabilization of the tubulin polymer was assessed by Western blotting for beta-tubulin distributions in supernatant and pellet fractions of cell lysates. A combination index (CI) was calculated from the equation CI = D1/Dx1 + D2/Dx2 in which D1 and D2 are the concentrations of drug 1 and drug 2 that in combination give the same response as drug 1 alone (Dx1) or drug 2 alone (Dx2). A CI of less than 1 indicates synergy, equal to 1, additivity, and greater than 1, antagonism. Confidence intervals for each CI value were obtained using a bootstrapping procedure. In cell proliferation assays, statistically significant synergy was found between peloruside A and paclitaxel and epothilone A. Combinations of these two taxoid site drugs, however, also showed synergy in their effects on cell proliferation. These results confirm that peloruside A, when added in combination with other microtubule stabilizing agents, acts synergistically to enhance the antimitotic action of the drugs, but also highlight the complexity of drug interactions in intact cells.