The thiocarbamate drug disulfiram has been used for decades in the treatment of alcohol abuse. Disulfiram induces apoptosis in a number of tumor cell lines and was recently by us proposed to act as a 26S proteasome inhibitor. In this work we characterized disulfiram in vitro with regard to tumor-type specificity, possible mechanisms of action and drug resistance and cell death in human tumor cell lines and in 78 samples of tumor cells from patients using the fluorometric microculture cytotoxicity assay and the automated fluorescence-imaging microscope ArrayScan((R)). Disulfiram induced cytotoxicity in a biphasic pattern in both cell lines and patient tumor cells. Disulfiram induced apoptosis as measured by cell membrane permeability, nuclear fragmentation/condensation and caspase-3/7 activation using high content screening assays. For many of the cell lines tested disulfiram was active in sub-micromolar concentrations. When comparing the logIC(50) patterns with other cytotoxic agents, disulfiram showed low correlation (R<0.5) with all drugs except lactacystin (R=0.69), a known proteasome inhibitor, indicating that the two substances may share mechanistic pathways. Disulfiram was more active in hematological than in solid tumor samples, but substantial activity was observed in carcinomas of the ovary and the breast and in non-small cell lung cancer. Disulfiram also displayed higher cytotoxic effect in cells from chronic lymphocytic leukemia than in normal lymphocytes (p<0.05), which may indicate some tumor selectivity. These results together with large clinical experience and relatively mild side effects encourage clinical studies of disulfiram as an anti-cancer agent.