The influence of extracellular matrix conditions and plating density on cell cytoarchitecture and the constitutive and chemically induced expression of cytochrome P450 3A4 (CYP3A4) was examined in primary cultures of human hepatocytes. Constitutive and drug-induced microsomal CYP3A4 expression occurred equally well in human hepatocyte cultures maintained on either a complex or simple substratum (Matrigel vs collagen, type I), or in a sandwich configuration (i.e., between two layers of extracellular matrix), despite the markedly different morphological properties exhibited by each condition. However, a density-dependent decrease in both the constitutive and induced levels of CYP3A4 was observed in hepatocytes maintained on a simple collagen substratum as plating density was reduced from 100% to 25%. Marked alterations in cell shape and cytoarchitecture were noted concomitant with decreases in the expression and localization of intercellular gap junctions and E-cadherin-mediated cell adhesions. In addition, the intracellular distribution of microtubules and microfilaments was altered substantially and the expression of immunoreactive actin and beta-tubulin increased as cell density was decreased. These effects were reversed to some extent by overlaying monolayers with extracellular matrix or by co-culturing with another cell type. Efforts to maintain normal cell shape and cytoskeletal distribution in hepatocytes at low cell density with a Matrigel substratum failed to restore normal basal levels of CYP3A4 expression or responsiveness to rifampicin (RIF). Likewise, E-cadherin and Cx-32 expression was again reduced, even though the distribution and expression of cytoskeletal elements returned to normal levels. These results suggest that cell-cell contacts, but not the extracellular matrix configuration or composition, play a critical role in determining normal responsiveness to chemical modulators in human hepatocytes.