BACKGROUND Although it has been known for more than three decades that administration of lipophilic chemicals, including phenobarbital, produces liver hypertrophy, proliferation of smooth endoplasmic reticulum, and induction of liver microsomal enzymes such as cytochromes P450 (CYP) 2B1 and 2B2, the mechanism of this adaptive response remains largely unknown. An important advance was the recognition that, unlike cultures of continuously proliferating liver cell lines or cultures of primary non-proliferating adult rat hepatocytes maintained on either plastic or collagen-coated dishes, hepatocytes cultured on a basement membrane gel, Matrigel, formed rounded clusters and permitted phenobarbital-mediated induction in vitro of CYP 2B1/2 mRNAs and immunoreactive proteins (1). RESULTS We cultured adult rat hepatocytes on Type 1 collagen (Vitrogen) and allowed the cells to spread, flatten, and firmly attach to the substratum. Subsequent incubation in medium containing Matrigel as a soluble component, fully restored, in a dose-dependent manner, the ability to respond to phenobarbital with induction of CYP 2B1/2 mRNAs. Repeating this experiment with medium containing equivalent amounts of purified laminin, a major component of Matrigel, or with YIGSR or SIKVAV, two peptides known to mimic various activities of laminin, similarly restored phenobarbital responsiveness to hepatocytes cultured on Vitrogen. In contrast, use of equal amounts of SHA-23, a scrambled peptide relevant to SIKVAV, produced no such effect. None of these treatments caused a rounding or any other observable change in the flattened, cellular morphology, making it unlikely that cell-spreading or alterations in cell shape account for loss of such differentiated liver functions as phenobarbital induction of CYP 2B1/2 mRNAs in cultured hepatocytes on Vitrogen. Hepatocytes cultured on Matrigel in the presence of either colchicine, cytochalasins B and D, nocodazole, or taxol did not show induction of 2B1/2 mRNAs by phenobarbital specifically, while the amounts of both albumin and glucose-6-phosphate dehydrogenase (G6PD) mRNAs were unaffected. CONCLUSIONS We conclude that the process by which phenobarbital induced 2B1/2 mRNAs in hepatocytes appears to require highly concerted effects of specific extracellular components prominently involving laminin. This likely occurs through a signal transduction process requiring probably both microfilament and microtubular integrity.