Cultured primary hepatocytes are one of the most suitable in vitro models for hepatic toxicological studies. Unfortunately, there is a temporal loss of liver-specific function in culture that limits their utility for some applications. Plating hepatocytes on a substratum has been shown to stabilize the differentiated phenotype for short-term culture. In order to identify the substratum that best supports in vivo basal hepatocyte gene expression profiles in vitro, the gene expression profiles of primary rat hepatocytes plated on collagen I in hepatocyte maintenance medium (HMM) or hepatocyte culture medium (HCM), or on matrigel in HMM medium for 2 h, 16 h, or 72 h were compared to the expression profiles of freshly isolated rat hepatocytes using the Atlas rat stress array. After 16 h in culture, there were differences in gene expression between cultured primary hepatocytes and freshly isolated hepatocytes, but no apparent substratum effects. At 72 h, the expression of 9 genes was altered in hepatocytes plated on either substratum compared to expression in freshly isolated hepatocytes. However, there were an additional 13 genes with increased expression in hepatocytes plated on collagen I that were expressed at low or non-detectable levels in freshly isolated hepatocytes or primary hepatocytes plated on matrigel. In summary, after 72 h, primary hepatocytes plated on matrigel had basal gene expression patterns more similar to patterns in freshly isolated hepatocytes than did hepatocytes cultured on collagen. In addition, culture on matrigel suppressed the expression of atypical genes in culture. These preliminary studies suggest that culture on matrigel may be preferable for longer-term in vitro toxicological studies.