At an early stage of drug discovery high throughput screens are an invaluable tool to de-select compounds with undesirable properties. A high throughout in vitro toxicity screen has been developed and validated to identify compounds that have a high potential to be acutely toxic in vivo. This screen is based on treating Chinese hamster ovary (CHO) cells with test compounds for 24 h and then determining the degree of cytotoxicity by the reduction of Resazurin. Twenty-six structurally unrelated compounds were chosen that spanned a range of acute LD(50) values and mechanisms of toxicity. The acute LD(50) values (intraperitoneal and intravenous routes) from rat and mouse were taken from the RTECS database. Experimentally derived in vitro IC(35) results were compared to the 'most toxic' (lowest) LD(50) values for each compound. The resulting correlation was statistically significant (r=0.8475). However, due to the scatter of the data points, it was considered not appropriate to rank compounds according to their degree of in vivo toxicity on the basis of the in vitro result. However, by defining cut-off concentrations for both the in vivo (LD(50)) and the in vitro (IC(35)) values it was possible, using the in vitro result (IC(35) <10 microM), to identify compounds that had a high potential to be acutely toxic in vivo ('most toxic' LD(50) <25 micromol/kg). Further development led to a high throughput screen capable of giving a 'Yes', 'No' or 'Borderline' classification as to whether a compound has a high acute in vivo toxic potential. This screen is highly specific (no false positive classifications) and has a sensitivity of approximately 80%. This is deemed acceptable for a first tier toxicity screen at an early stage in the drug discovery process. Transfer of this screen from GlaxoSmithKline UK to sites in Italy, Spain and the USA resulted in very similar findings indicating the inter-laboratory robustness of this screen and therefore the ability to compare results across the GlaxoSmithKline sites.