Mimicking the in vivo microenvironment is one of the current strategies to maintain liver-specific functionality in primary cultured hepatocytes for long periods. Freshly isolated hepatocytes entrapped in collagen gel type I (collagen gel immobilization culture) or sandwiched between two layers of hydrated collagen type I (collagen gel sandwich culture) are known to display liver-specific functions (e.g., biotransformation capacity) for more than 6 wk. We describe how to set up both types of organotypical hepatocyte culture systems. Besides a detailed protocol, we give some practical tips, taken from our own experience with long-term hepatocyte culture.