PEA‐15 (phosphoprotein enriched in astrocytes, M_r = 15,000) is an acidic serine‐phosphorylated protein highly expressed in the CNS, where it can play a protective role against cytokine‐induced apoptosis. PEA‐15 is a major substrate for protein kinase C. Endothelins, which are known to exert pleiotropic effects on astrocytes, were used to analyze further the processes involved in PEA‐15 phosphorylation. Endothelin‐1 or endothelin‐3 (0.1 µM) induced a robust phosphorylation of PEA‐15 that was abolished by the removal of extracellular calcium, but only diminished by inhibitors of protein kinase C. Microsequencing of phosphopeptides generated by digestion of PEA‐15 following endothelin‐1 treatment identified two phosphorylated residues: Ser¹⁰⁴, previously recognized as the protein kinase C site, and a novel phosphoserine, Ser¹¹⁶, located in a consensus motif for either protein kinase casein kinase II or calcium/calmodulin‐dependent protein kinase II (CaMKII). Partly purified PEA‐15 was a substrate in vitro for CaMKII, but not for casein kinase II. Two‐dimensional phosphopeptide mapping demonstrated that the site phosphorylated in vitro by CaMKII was also phosphorylated in intact astrocytes in response to endothelin. CaMKII phosphorylated selectively Ser¹¹⁶ and had no effect on Ser¹⁰⁴, but in vitro phosphorylation by CaMKII appeared to facilitate further phosphorylation by protein kinase C. Treatment of intact astrocytes with okadaic acid enhanced the phosphorylation of the CaMKII site. These results demonstrate that PEA‐15 is phosphorylated in astrocytes by CaMKII (or a related kinase) and by protein kinase C in response to endothelin.