Abstract: PEA-15 has recently been identified as a major phosphoprotein in astrocytes and an endogenous substrate for protein kinase C. This 15-kDa protein exists under three molecular forms, an unphosphorylated form, N, and two phosphorylated forms, Pa and Pb. Ȧntisera were raised against synthetic peptides corresponding to the internal sequences of the mouse protein containing the two specific phosphorylation sites and affinity-purified antibodies were used for immunoblotting. PEA-15 was found mainly in the cytosol, but its protein kinase C-phosphorylated form, Pb, was also detectable in association with the membrane and remained with the fraction that contains stabilized microtubules. Abundant in astrocytes, particularly in the hippocampus, PEA-15 was also detected in all cultured brain cell types examined, indicating a more ubiquitous distribution of the protein, further demonstrated by its detection in the eye and in the lung. Parallel to the increase in expression levels, phosphorylation of PEA-15 also increased during development. This paralleled results obtained in primary cultures, where PEA-15 levels increase with cell maturation. Finally, physiological importance of PEA-15 phosphorylation was illustrated by immunoreactivity observed in brain homogenates of different mammals, birds, amphibians, and fish.