We have analyzed the biochemical defect in a mutant line of mice that produces less than 1% of the normal level of serum transferrin. This mouse line (Hp) transcribes the transferrin gene in liver at the same rate observed in normal mice, but the steady state levels of transferrin mRNA sequences are less than 20% of normal. Further hybridization studies reveal that most of the transferrin mRNA sequences present in homozygous Hp mouse liver are in the form of a 5 kb nuclear precursor instead of the mature 2.5 kb transferrin mRNA seen in normal mice. Using several different exon and intron probes from the mouse transferrin gene, we have shown that the 5 kb RNA precursor retains the last two introns of the transferrin gene but that the 5′ and middle introns have been removed by processing. The defect in transferrin mRNA processing also extends to nonhepatic tissues and we find the same lack of mature mRNA and increased precursor accumulation in brain RNA. Since Southern blot analysis does not reveal gross changes in the structure of the transferrin gene in Hp mice, we suggest that the Hp defect is due to a small deletion or point mutation that either disrupts splicing signals or uncovers cryptic splice signals that interfere with processing of the last two introns in the transferrin gene. This Hp mouse line provides an opportunity to study the effects of transferrin deficiency on development and iron homeostasis.