Toxicological studies using primary cultured hepatocytes were reviewed. Primary cultures of mature hepatocytes retain many liver functions and various hormonal responses for long term. Therefore, this system overcomes various limitations of another in vitro system using liver. Toxicological studies using primary cultured hepatocytes showed high correlation to in vivo studies, for example, in vitro screening of inducible chemicals of hepatitis, and unscheduled DNA synthesis activity, etc. But there still remain some shortcomings, for example, rapid loss of cytochrome P450 and other drug metabolizing enzymes. A number of attempts to modify culture conditions resulted in long term maintenance of these enzymes. Recently Guzelian and colleagues established that using matrigel coated dishes, primary cultured hepatocytes expressed highly and maintained cytochrome P450 and other specific genes for long periods like in vivo. In the future study using cultured hepatocytes, if it can culture more long term or subculture as maintenance liver functions, not only rodent hepatocytes but also human hepatocytes are more useful for toxicological assessments and screening of drugs for liver diseases.