Phosphorylation of chloroplast thylakoid proteins, in particular light harvesting complex II (LHC II), is believed to play an important role in regulating photosynthetic electron transfer. Evidence supporting the involvement of multiple protein kinases in this system is mounting. We have re-examined pea thylakoid membranes and found evidence for a membrane-associated protein tyrosine kinase (PTK). Phosphorylation of many thylakoid proteins, including LHC II, is sensitive to treatment with the tyrosine kinase inhibitor genistein. Anti-phosphotyrosine antibodies react specifically with nine thylakoid proteins, two of which have been identified as components of LHC II. The phosphate associated with these two proteins is also resistant to strong base and acid treatment, further substantiating the assignment of phosphotyrosine. Potential interactions between this novel chloroplast PTK activity and the well-documented threonine kinase activities are discussed and the presence of a cascade of thylakoid protein kinases is proposed.