Previous studies have indicated that high levels of insulin synthesis occur in the yolk sac of fetal rats. Because the yolk sac is an early site for synthesis of several tissue-specific proteins synthesized by liver later in development, these studies were performed to determine whether insulin gene expression also occurs in fetal liver. To this purpose, liver RNA obtained on consecutive days of rat fetal development from embryo day (E) 13 to E21 was evaluated for the presence of insulin or insulin-like mRNA species using Northern hybridization with a uniformly labelled rat insulin II genomic antisense RNA probe. Two species were detected. The larger was approximately 2.4 kilobases in length, was very low in abundance, and was present only during the earliest days studied (E13-15). The second species was approximately 720 bases in length, increased in abundance between days E13-16, and decreased between days E16-21. Maximum abundance of this mRNA was 0.3 µg/ng total liver RNA, or 1/10th to 1/20th the abundance of total insulin mRNA in adult rat pancreas. Sequencing of multiple cloned products of E15 rat liver cDNA amplified by polymerase chain reaction using insulin I or II gene-specific primers indicated that the bands detected on Northern hybridization were (ancestral) rat insulin II gene transcripts. Analysis of products of polymerase chain reactions also indicated that the duplicated rat insulin I gene was not expressed in fetal liver. The content of insulin mRNA in fetal liver is sufficient to suggest that the liver may be a significant source for insulin at specific times during fetal development.