We have sequenced rabbit cDNAs that encode one isoform of the alpha subunit and two isoforms of the beta subunit of phosphorylase kinase, in addition to the single isoform from fast skeletal muscle that has been characterized to date for each subunit. All these isoforms are generated by alternative RNA splicing. The alpha subunit sequence obtained from slow skeletal muscle (soleus) is characterized by an internal deletion of 59 amino acids. This deletion is predominant in mRNA from slow muscle, heart, and uterus and accounts for the smaller alpha subunit variant (alpha') characteristic of phosphorylase kinase purified from slow muscle and heart. The beta subunit mRNA can be differentially spliced at two sites. In all tissues (except skeletal muscle) that were analyzed, an internal segment encoding 28 amino acids of the muscle sequence is replaced by a homologous sequence of identical length, presumably through the use of mutually exclusive exons. In brain and some other tissues, the deduced N-terminal sequence of the beta subunit is also changed. This is achieved by an insertion into the mRNA sequence that interrupts the initial reading frame after 25 codons and starts a new reading frame, encoding a different N terminus of 18 amino acids. This modification probably affects the major regulatory phosphorylation site of the beta subunit.