Many different cancer types have previously been found to show increased uptake of the vitamins folate, vitamin B12, and biotin; however, it is not known whether these tumor lines show increased uptake of one or more of the vitamins. The current study was designed to examine the relative uptake of the three vitamins in 10 different types of cell lines. Rhodamine-labeled hydroxypropyl-methacrylamide (HPMA) was targeted with vitamin B(12), folate, or biotin, and the uptake of the labeled polymer was compared both in in vitro cell cultures and in mice-bearing tumors from a variety of tumor cell lines. Fluorescent microscopy of cell cultures and histological examination of tumor sections showed greatly increased uptake of the fluorescently labeled polymer in many tumors when the polymer was targeted with folate, biotin, or vitamin B(12). Tumors with enhanced uptake of vitamin B(12)- or folate-targeted rhodamine-HPMA also showed increased uptake of biotin-Rho-HPMA. In contrast, tumors with increased uptake of folate-Rho-HPMA did not show increased uptake of vitamin B12 (VB(12))-HPMA and vice versa. These findings suggest that vitamin-targeted polymers may greatly increase the uptake of drug-polymer complexes in certain tumors, which may result in an increased efficacy of antitumor agents, and which may allow for easier imaging of both the primary and metastatic tumors.