PYGB is a new isozyme of glycogen phosphorylase (1,4-D-glucan:orthosphosphate D-glucosyltransferase; EC 2.4.1.1) from a cDNA library prepared from a human brain astrocytoma cell line. Blot-hybridization analysis showed that the message is preferentially expressed in human brain, but is also found at a low level in human fetal liver and adult liver and muscle tissues. The protein sequence deduced from the nucleotide sequence was 862 amino acids long compared with 846 and 841 amino acids for the liver (PYGL) and muscle (PYGM) isozymes, respectively. The greater length of brain phosphorylase was found to be due entirely to an extension at the c-terminal portion of the protein. Muscle and brain isozymes shared greater similarities with each other than either did with the liver sequence.