De novo formation of laminin-positive basement membranes was found to be a distinct morphologic feature of diethylnitrosamine/phenobarbital-induced hepatocellular carcinomas of the rat. The first appearance of extracellularly located laminin occurred in the preneoplastic liver lesions (corresponding to neoplastic nodules), and this feature became successively more prominent during the course of hepatocellular carcinoma development. Most groups of tumor cells were surrounded by laminin-positive basement membrane material. The laminin-positive material was also deposited along the sinusoids, a location where no laminin was seen in normal rat liver. The amount of extractable laminin from hepatocellular carcinomas was significantly higher (approximately 100 ng per mg tissue) than that of normal liver tissue (less than 20 ng per mg). In vitro experiments demonstrated that normal and preneoplastic rat hepatocytes had the capacity to lay down basement membrane-like material. This occurred, however, only when the hepatocytes were cocultured with certain feeder cells or when grown in the presence of their conditioned media. These results indicate that during experimental hepatocarcinogenesis in the rat some as yet undefined humoral factor(s) might influence the hepatocytes to turn on genes encoding the basement membrane components and further stimulate the assembly and deposition of basement membranes.