Transferrin (Tf), an iron-binding protein, was investigated in the cultured human fibroblast which is a major cell type in connective tissues. Tf is a major iron-transporting protein and has an important role in iron metabolism. Using two-dimensional gel electrophoresis, we demonstrated the eight subtypes of Tf produced by cultured normal human fibroblasts and their down-regulation after the immortalization of human cells, an essential early step of in vitro transformation. However, the amount of Tf mRNA in the immortalized cells was equal to that in the normal human fibroblasts, suggesting that the down-regulation occurred at the posttranscriptional level. The amount of Tf receptor increased in the immortalized cells in spite of a decrease in the amount of intracellular Tf. Interestingly the produced Tf associated with microtubules. These findings suggest a novel aspect of Tf characteristics in human fibroblasts.