The complete nucleotide sequence of ovine β-casein mRNA has been determined by sequencing, according to Sanger-Messing, both a recombinant clone isolated from a mammary cDNA pUC 18 library and a single-stranded cDNA generated by reverse transcription from a synthetic 17-mer primer complementary to the 5′ part of the mRNA coding frame. The 1088 nucleotide long β-casein mRNA, excluding the poly(A) tail, contains a coding frame of 669 nucleotides including the stop codon, flanked by 60 adn 359 nucleotides in the 5′ and 3′ untranslated regions, respectively. It arises from the splicing of 9 exons as deduced from gene sequence data. The deduced amino acid sequence differs at 3 positions from that previously determined by direct sequencing of mature β-casein. Comparison of the ovine, bovine, rat, mouse, and rabbit β-casein mRNA sequences shows a higher homology in the 3′ and 5′ untranslated regions. The most conserved regions in the open reading frame are essentially those encoding the signal peptide and the major phosphorylation site.