A region approximately 35 kilobase pairs (kb) in length containing the hormonally regulated rat gamma-casein gene has been characterized by examining overlapping clones of genomic rat DNA obtained from two Charon 4A libraries. The entire gamma-casein structural gene is contained in a single 17-kb phage clone. R-loop and restriction enzyme mapping analyses revealed that the gamma-casein gene is approximately 15 kb long and is, therefore, 17.4 times larger than the mature gamma-casein mRNA. The coding regions of the gamma-casein gene are split into at least nine small segments, interspersed with long intervening sequences. Sequence analysis of the 5' end of the gamma-casein gene revealed the presence of a TATA sequence which may play a role in the initiation of gene transcription. The first exon is 44 nucleotides long and encodes part of the 5' noncoding sequences of the gamma-casein mRNA. The first intron was found to contain a short interspersed repeated DNA sequences which shares a 92% homology with a cloned rat repeated DNA sequence found at the 3' end of several other rat genes. In addition, the gamma-casein gene contains several families of highly repeated sequences interspersed throughout the intervening and flanking regions, including a family of evolutionary conserved repeats. Thus, the gamma-casein gene represents an unusually large and complex split mammalian gene.