Rats raised on a low-iron diet were used as a model system for investigating the regulation of transferrin gene expression by iron deficiency. We quantitated transferrin mRNA in a variety of tissues from normal and iron-deficient rats and found that the level of transferrin mRNA in normal rat liver was about 6500 molecules per cell, while the level in iron-deficient animals was 2.4-fold higher. The increase of transferrin mRNA in iron deficiency was the result of a specific induction of transferrin gene transcriptional activity as measured in isolated nuclei. This increase in transferrin gene expression resulted in a corresponding increase in serum total-iron-binding capacity. Of the other tissues examined, moderate amounts of transferrin mRNA were found in brain (83 molecules per cell) and testis (114 molecules per cell), and low levels were measured in spleen and kidney. The transferrin mRNA content of brain, testis, spleen, and kidney remained unchanged in iron deficiency. The small intestine had no detectable transferrin mRNA in either normal or iron-deficient rats; however, transferrin protein was present, and its level was 2-fold higher in the iron-deficient group. We hypothesize that intestinal transferrin is synthesized in the liver and is delivered to the gut via the bile. Consistent with this idea, bile transferrin content was found to be elevated in iron deficiency and appeared to be sufficient to account for intestinal transferrin levels. In addition, treatment of plasma transferrin with bile caused an acidic shift in its isoelectric-focusing behavior so that it comigrated with intestinal transferrin.