Reversed-phase liquid chromatography (RPLC) was interfaced to mass spectrometry (MS) with an electrospray ion (ESI) source for the separation and accurate molecular mass determination of the individual intrinsic membrane proteins that comprise the photosystem II (PS II) major light-harvesting complex (LHC II) and minor (CP24, CP26 and CP29) antenna system, whose molecular masses range between 22,000 and 29,000. PS II is a supramolecular complex intrinsic of the thylacoid membrane, which plays the important role in photosynthesis of capturing solar energy, and transferring it to photochemical reaction centers where energy conversion occurs. The protein components of the PS II major and minor antenna systems were extracted from spinach thylacoid membranes and separated using a butyl-silica column eluted by an acetonitrile gradient in 0.05% (v/v) aqueous trifluoroacetic acid. On-line electrospray MS allowed accurate molecular mass determination and identification of the protein components of PS II major and minor antenna system. The proposed RPLC-ESI-MS method holds several advantages over sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the conventional technique for studying membrane proteins, including a better protein separation, mass accuracy, speed and efficiency.