There is considerable interest in the characterization of novel tumor-associated antigens that lend themselves to antibody-mediated pharmacodelivery strategies. Delta-like 1 homolog protein (DLK1), which exists both as transmembrane protein and in soluble form, shows a restricted pattern of expression in healthy organs, while being overexpressed in some tumors. We have generated a human antibody specific to DLK1 using phage display technology. This reagent was used for a comprehensive characterization of DLK1 expression in freshly frozen sections of normal human adult tissues and of xenografted human tumors. DLK1 was virtually undetectable in most organs, except for placenta which was weakly positive. By contrast, DLK1 exhibited a moderate-to-strong expression in 8/9 tumor types tested. Our analysis shed light on previous conflicting reports on DLK1 expression in health and disease. The study suggests that DLK1 may be considered as a target for antibody-mediated pharmacodelivery strategies, in view of the protein’s limited expression in normal tissues and its abundance in the interstitium of neoplastic lesions.