Wheat (Triticum aestivum L.) germ Ca2+-dependent protein kinase (CDPK) phosphorylates a wheat basic protein (WBP) which is a putative phospholipid transfer protein and one of the best protein substrates yet found for wheat CDPK. Reversed phase high performance liquid chromatography (HPLC) of the tryptic digest of [P-32]phospho WBP resolved two [P-32]phosphopeptide peaks. Edman sequencing of one of these established that WBP is phosphorylated by CDPK on S17 within the sequence (APCISYA)-P-13. WBP may also be phosphorylated at an additional site. Wheat CDPK also phosphorylates barley (Hordeum. vulgare) lipid transfer protein (LTP1) and [P-32]phosphoLTP1 exactly comigrates with LTP1 on SDS-PAGE. Unlike wheat WBP, LTP1 is a relatively poor substrate for wheat CDPK and is phosphorylated largely on threonine. Reversed phase high performance liquid chromatography (HPLC) of the tryptic digest of [P-32]phosphopeptide resolved one [P-32]phosphopeptide and subsequent Edman sequencing of this phosphopeptide showed that LTP1 is phosphorylated on T15 within the sequence (KPCLTYVQ)-P-11. This phosphorylation site amino acid sequence motif differs from the Basic-X-X-Ser(Thr) motif found for a variety of synthetic peptide substrates of wheat germ CDPK but corresponds to the Pro-Cys-X-Ser/Thr phosphorylation site motif found for the phosphorylation by wheat germ CDPK of WBP, which is a homologue of barley LTP1. The phosphorylation sites determined for WBP and LTP1 are similar structurally and are within precisely aligned homologous sequences.