We investigated the relationship between the expression of tyrosine aminotransferase (TAT) and cytoskeletal systems of cultured rat hepatocytes by using serum-free culture conditions and changing three factors: 1) the concentration of calcium, 2) the dish-coating material, and 3) the cell-plating density. In hepatocytes in low-calcium medium, induction of TAT by dexamethasone and glucagon was maintained, although cell-cell adhesion was lost. Hepatocytes on Matrigel formed a nonspreading, spherical shape that provided them with the full extent of TAT activity without cell-cell adhesion. Hepatocytes plated on collagen at low cell density spread and changed shape, and the induction of TAT activity was markedly reduced. By using confocal laser-scanning microscopy, we analyzed the three-dimensional organization of cytoplasmic microtubules of hepatocytes maintaining the ability of TAT induction. Hepatocytes plated on collagen at low cell density possessed the radial filamentous structure of cytoplasmic microtubules. When the spherical shape of hepatocytes was maintained by cultivating cells on Matrigel, a ring-like structure of cytoplasmic microtubules beneath the plasma membrane was dominant. Moreover, the induction of TAT activity of hepatocytes in a standard culture system was strongly inhibited by the addition of 1 microM colchicine. These studies suggest that the organization of cytoplasmic microtubules may participate in the shape-related regulation of cell function.