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Abstract: The objective of this study was to explain the male sterile mechanism of wheat (Triticum aestivum L.) based on chloroplast proteome. A method for isolating intact chloroplast proteome from wheat floret was established. Using this method, the chloroplast proteomes were extracted from florets of the genic and physiological male sterile lines and their maintainer line, and separated in 2-dimensional electrophoresis (2-DE) gels. The cytoplasmic-nuclear sterile line, ms(S)-1376, had identical nuclear background with the maintainer line, (A)-1376, and the physiological male sterile line, ms(A)-1376, was derived from (A)-1376 after induction of chemical hybridizing agent SQ-1. The extraction method was effective to obtain high purity of intact chloroplast using discontinuous sucrose density gradient centrifugation with 3-step gradient densities of 30, 45, and 60%. The 2-DE result showed that the floret chloroplast protein profiles were different among the 3 lines at uninucleate anther stage, and 239 protein spots were visible on each gel (pH 4-7, molecular weight 14.4-66.2 kD). Six differentially expressed proteins were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) and indexed in bioinformation database. They were identified as acyl-CoA dehydrogenase domain protein, calmodulin-binding protein phosphatase, multiple catalytic peptidase, heat-shock protein 60, light receptor protein 2, and a protein of unknown function. These proteins are involved in series of physiological reactions such as metabolism of energy substances, chloroplast defendance, chloroplasts signal transduction, and plant growth. The differential expressions of these proteins among the 3 lines are likely related to the male sterility in wheat. [Copyright &y& Elsevier]