Male reproductive development in rice is very sensitive to various forms of environmental stresses including low temperature. A few days of cold treatment (<20 degrees C) at the young microspore stage induce severe pollen sterility and thus large grain yield reductions. To investigate this phenomenon, anther proteins at the early stages of microspore development, with or without cold treatment at 12 degrees C, were extracted, separated by two-dimensional gel electrophoresis, and compared. The cold-sensitive cultivar Doongara and the relatively cold-tolerant cultivar HSC55 were used. The abundance of 37 anther proteins was changed more than 2-fold after 1, 2, and 4 days of cold treatment in cv. Doongara. Among them, one protein was newly induced, 32 protein spots were up-regulated, and four protein spots were down-regulated. Of these 37 protein spots, we identified two anther-specific proteins (putative lipid transfer protein and Osg6B) and a calreticulin that were down-regulated and a cystine synthase, a beta-6 subunit of the 20 S proteasome, an H protein of the glycine cleavage system, cytochrome c oxidase subunit VB, an osmotin protein homologue, a putative 6-phosphogluconolactonase, a putative adenylate kinase, a putative cysteine proteinase inhibitor, ribosomal protein S12E, a caffeoyl-CoA O-methyltransferase, and a monodehydroascorbate reductase that were up-regulated. Identification of these proteins is available upon request. Accumulation of these proteins did not vary greatly after cold treatment in panicles of cv. Doongara or in the anthers of the cv. HSC55. The newly induced protein named Oryza sativa cold-induced anther protein (OsCIA) was identified as an unknown protein. The OsCIA protein was detected in panicles, leaves, and seedling tissues under normal growth conditions. Quantitative real time RT-PCR analysis of OsCIA mRNA expression showed no significant change between low temperature-treated and untreated plants. A possible regulatory role for the newly induced protein is proposed.