Four chemicals that had been tested in an in vivo toxicological screen were tested in a Chinese hamster ovary (CHO) cytotoxicity assay. Cell density, viability, ATP concentration, rate of protein synthesis, and cellular protein concentration were decreased by exposure to acrylamide (AC), chlordecone (CHL), cyclophosphamide (CYC), and diethylstilbestrol (DES). Based on the in vitro toxicity rankings, DES and CHL were more toxic than AC or CYC. The ability of the CHO assay to respond to DES and CHL was comparable to other published in vitro assays. However, the CHO assay was unresponsive to concentrations of AC and CYC detected by assay systems composed of cells derived from the nervous system and assays containing metabolic activation, respectively. In vivo, the four chemicals were toxic following 10 daily treatments. While CYC was the least toxic chemical in vitro, it was one of the most toxic in vivo. This lack of correlation between the in vivo and the in vitro data in the study may be due to the lack of metabolic activation in the CHO cytotoxicity assay or may indicate the inability of the CHO cytotoxicity assay to discriminate between highly toxic chemicals.