In most cases, cytotoxic drugs do not preferentially accumulate at the tumor site, causing unwanted toxicities and preventing dose escalation to therapeutically active regimens. Here, we show that acetazolamide derivatives, which bind to carbonic anhydrase IX (CAIX) on the surface of kidney cancer cells, selectively deliver payloads at the site of disease, sparing normal organs. Biodistribution studies, performed in tumor-bearing mice with acetazolamide derivatives bearing a technetium-99m chelator complex or a red fluorophore as payload, revealed a preferential tumor accumulation of the compound at doses up to 560 nmol/Kg. The percentage of injected dose per gram in the tumor was dose-dependent and revealed optimal tumor:organ ratios at 140 nmol/Kg, with a tumor:blood ratio of 80:1 at 6 h. Acetazolamide, coupled to potent cytotoxic drugs via a dipeptide linker, exhibited a potent antitumor activity in nude mice bearing SKRC-52 renal cell carcinomas, while drug derivatives devoid of the acetazolamide moiety did not exhibit any detectable anticancer activity at the same doses. The observation of tumor regression with a noninternalizing ligand and with different cytotoxic moieties (MMAE and PNU-159682) indicates a general mechanism of action, based on the selective accumulation of the product on tumor cells, followed by the extracellular proteolytic release of the cytotoxic payload at the neoplastic site and the subsequent drug internalization into tumor cells. Acetazolamide-based drug conjugates may represent a promising class of targeted agents for the treatment of metastatic kidney cancer, as the majority of human clear cell renal cell carcinomas are strongly positive for CAIX.