PepT1, which is localized in the small intestinal and renal epithelial brush border membranes, takes up native di and tripeptide as well as peptidemimetic drugs such as β-lactam antibiotics and bestatin. The limited tissue distribution and broad specificity of PepT1 may be advantageous to deliver peptidemimetic drugs to the tissues which need to be supplied of nutrients for continuous growth. Accordingly, we investigated the substrate specificity of human PepT1 and measured the oligopeptide transport activities in tumor cells using human fibrosarcoma cell line, HT-1080 as the model tumor cells. Although it has been unclear whether cefdinir and cefaclor are transported by PepT1 or not, the present study clearly showed that they are transported by PepT1. The affinity of zwitterionic antibiotics to PepT1 is maximal around pH 6, but in anionic ones, higher activity was observed at more acidic pH. These results indicate that PepT1 has a broad specificity and its transport activity is influenced by ionization state of substrates. To explore the feasibility of targeting human tumor cells via the peptide transport system, oligopeptide uptake was studied in HT-1080 and compared with that in normal the human fibroblast cell line IMR-90 by measuring the uptake of radiolabeled Gly-Sar. The uptake of Gly-Sar by HT-1080 was concentration and pH dependent. The initial rate of Gly-Sar uptake had a Km of 11.4 mM and Vmax of 26.8 nmol/15 min/mg protein and was inhibited by various di and tripeptides, but not by amino acids, tetra or pentapeptides. On the other hand, IMR-90 showed negligible peptide transport activity. The present finding could be the basis of a novel strategy for the specific delivery of oligopeptide-mimetic anticancer drugs into tumor cells.