Multiple forms of acetyl-CoA carboxylase mRNA were previously detected in the mammary gland (Lopez-Casillas, F., Luo, X., Kong, I.-S., and Kim, K.-H. (1989) Gene, in press). We have now established that the rat liver also contains heterogeneous acetyl-CoA carboxylase mRNA populations that differ in the 5'-untranslated region. In addition, the liver contains a unique form of acetyl-CoA carboxylase mRNA in which the 5'-nontranslated end differs from the species in mammary gland. The 5' end of this unique species was characterized using a procedure for cloning minute amounts of primer extension products (pAU clones). This procedure should also be useful for obtaining full length clones of other mRNAs. The DNA sequence of pAU clones indicates that this liver-specific acetyl-CoA carboxylase mRNA has a 315-base long untranslated region. The first 242 nucleotides replace the 5' end of the predominant acetyl-CoA carboxylase mRNA found in the mammary gland (FL56 type). Under lipogenic conditions the unique liver acetyl-CoA carboxylase mRNA increases and is the major species of acetyl-CoA carboxylase mRNA. Livers from rats fed a normal diet and the mammary glands of lactating rats do not contain detectable amounts of the pAU type mRNA. On the other hand, the epididymal adipose tissue from these animals contains mainly the pAU type and only minimal amounts of the FL56 type. The multiple forms of acetyl-CoA carboxylase mRNA appear to be generated by differential splicing. In addition, transcription appears to be physiologically regulated by the use of tissue-specific acetyl-CoA carboxylase gene promoters.