The dominant male sterility gene Ms2 in wheat has been widely used in recurrent selection and variety improvement. Identification of genes associated with the male sterility in Ms2-carrying wheat will help us understand how Ms2 functions. Using a pair of isogenic lines of Ms2, subtractive hybridization was conducted with cDNA from bulked spikelets at meiophase of sterile plants as the tester and cDNA from the same tissues of fertile plants as the driver. Two major bands at 270 bp and 450 bp were obtained by suppression PCR (polymerase chain reaction) of the subtractive cDNA. A total of 882 recombinants from PCR product cloning were isolated for reverse Northern analysis. The results demonstrated that up to 90% of the inserts in the library were up-regulated in the sterile spikelets. Twenty-one unique inserts from this library were sequenced. Similarity search showed that eighteen of them were homologous to ESTs (expression sequence tags) derived from spike or anther tissues at meiophase. The chromosome locations of nine of the ESTs were determined using C.S. (Chinese spring) nulli-tetrasomic lines, one of which was assigned to chromosome group 4 that includes chromosome 4D where Ms2 is located. In addition, four additional ESTs could also be assigned to this group according to their homology to BACs (bacterial artificial chromosomes) or PAC (P1 artificial chromosomes) of rice chromosome 3. The expression patterns of eight of the inserts examined displayed increased expression in spikelets and anthers of the sterile plants.