To develop and validate a practical, in vitro, cell-based model to assess human hepatotoxicity potential of drugs, we used the new technology of high content screening (HCS) and a novel combination of critical model features, including (1) use of live, human hepatocytes with drug metabolism capability, (2) preincubation of cells for 3 days with drugs at a range of concentrations up to at least 30 times the efficacious concentration or 100 microM, (3) measurement of multiple parameters that were (4) morphological and biochemical, (5) indicative of prelethal cytotoxic effects, (6) representative of different mechanisms of toxicity, (7) at the single cell level and (8) amenable to rapid throughput. HCS is based on automated epifluorescence microscopy and image analysis of cells in a microtiter plate format. The assay was applied to HepG2 human hepatocytes cultured in 96-well plates and loaded with four fluorescent dyes for: calcium (Fluo-4 AM), mitochondrial membrane potential (TMRM), DNA content (Hoechst 33,342) to determine nuclear area and cell number and plasma membrane permeability (TOTO-3). Assay results were compared with those from 7 conventional, in vitro cytotoxicity assays that were applied to 611 compounds and shown to have low sensitivity (<25%), although high specificity ( approximately 90%) for detection of toxic drugs. For 243 drugs with varying degrees of toxicity, the HCS, sublethal, cytotoxicity assay had a sensitivity of 93% and specificity of 98%. Drugs testing positive that did not cause hepatotoxicity produced other serious, human organ toxicities. For 201 positive assay results, 86% drugs affected cell number, 70% affected nuclear area and mitochondrial membrane potential and 45% affected membrane permeability and 41% intracellular calcium concentration. Cell number was the first parameter affected for 56% of these drugs, nuclear area for 34% and mitochondrial membrane potential for 29% and membrane permeability for 7% and intracellular calcium for 10%. Hormesis occurred for 48% of all drugs with positive response, for 26% of mitochondrial and 34% nuclear area changes and 12% of cell number changes. Pattern of change was dependent on the class of drug and mechanism of toxicity. The ratio of concentrations for in vitro cytotoxicity to maximal efficaciousness in humans was not different across groups (12+/-22). Human toxicity potential was detected with 80% sensitivity and 90% specificity at a concentration of 30x the maximal efficacious concentration or 100 microM when efficaciousness was not considered. We conclude that human hepatotoxicity is highly concordant with in vitro cytotoxicity in this novel model and as detected by HCS.