Synthetic oligonucleotides were designed to amplify DNA sequences related to the heat-stable protein kinase inhibitor (PKI) isolated from rabbit skeletal muscle. Using these oligonucleotides, a 167-base pair fragment was isolated and shown to code for a portion of the mouse protein kinase inhibitor gene. This amplified DNA sequence was used to isolate three cDNA clones from a mouse brain cDNA library. A composite sequence was derived from these clones and contained a 228-nucleotide open reading frame encoding a protein of 76 amino acids. In addition, the sequence contained 29 nucleotides of 5'-untranslated and 2022 nucleotides of 3'-untranslated regions of the mouse PKI mRNA. Northern blot analysis of various mouse tissues indicated that the 3.8-kilobase pair mRNA is present at high levels in skeletal muscle and brain but is present at lower levels in heart, testis, and liver. RNase protection experiments also suggested that skeletal muscle and brain represent tissues of highest expression and that similar nucleotide sequences are found in the skeletal muscle, brain, and testicular transcripts. Southern blot analysis indicated a single prominent species of genomic DNA sequence related to the mouse PKI cDNA clones but a minor species was also detected.