Antitumor activity of linear, beta-cyclodextrin polymer (CDP)-camptothecin (CPT) conjugates (HGGG6, LGGG10, HG6, and HGGG10) is investigated in nude mice bearing human LS174T colon carcinoma tumors. These conjugates differ in polymer molecular mass [97 kDa (H) or 35 kDa (L)], CDP-CPT linker structure [glycine (G) or triglycine (GGG)], and CPT loading [ca. 6 wt % (6) or 10 wt % (10)]. Maximum tolerable doses (MTDs) of the three conjugates, LGGG10, HG6, and HGGG10, are determined to be 36, 9, and 9 mg of CPT/kg, respectively, while the MTD of the CDP alone exceeds 240 mg/kg (highest value investigated). The three CDP-CPT conjugates with high polymer molecular masses (HGGG6, HG6, and HGGG10) demonstrate antitumor activity at their MTDs superior to that of CPT at the same amount and to that of irinotecan at its optimal dose. They also show tumor growth inhibition that is superior to that of the conjugate containing the low-molecular mass polymer (LGGG10) at the same dose of CPT. No significant effects of CPT weight loading or linker structure on tumor growth delay are observed. However, conjugates containing G appear to be less toxic than these with GGG. These antitumor studies demonstrate that the CDP-based conjugates of CPT exhibit tumor growth inhibition superior to that of CPT or irinotecan at the conditions employed in this study. The striking observation is that a short course of treatment with the polymer conjugates gives long-term control of tumor growth that does not occur with either CPT or irinotecan. Intracellular CDPs are demonstrated by analyzing cells that were cultured in the presence of rhodamine-labeled CDP (HRhod) containing medium using both confocal microscopy and flow cytometry. The long-term therapeutic efficacy of CDP-CPT conjugates observed in mice may in part be due to the sustained release of CPT from these conjugates in the acidic, intracellular compartments since these conjugates are shown to have significantly slower release rates at acidic pH than at physiological pH.