Nodulin 26 is a nodule-specific protein that is associated with the symbiosome membrane of soybean root nodules. Nodulin 26 is an endogenous substrate for a novel calcium-dependent protein kinase (CDPK) of soybean root nodules. By phosphopeptide mapping of endoproteinase Lys-C-digested nodulin 26 and automated and manual peptide sequence analyses, we have identified the site on nodulin 26 phosphorylated by CDPK. We have also established that the phosphorylation site of nodulin 26 is identical to the phosphorylation site of CK-15, a synthetic peptide with the carboxyl-terminal sequence of nodulin 26. The phosphorylation of nodulin 26 occurs at position Ser262, and the phosphorylation of CK-15 occurs at the analogous position, Ser,6 in vitro. Thus, the CK-15 sequence apparently contains sufficient structural features of the phosphorylation site of nodulin 26 to be recognized by CDPK. On the basis of peptide mapping analysis of nodulin 26 from nodules that are metabolically labeled with [32P]phosphate, it appears that the site of nodulin 26 that is phosphorylated in vitro is also labeled in vivo. The data indicate that the carboxyl terminus of nodulin 26 is phosphorylated by CDPK and provide initial sequence data for the phosphorylation site of an endogenous substrate for a plant CDPK.