A cDNA subtraction and differential hybridization strategy was used to isolate cDNAs expressed early during male gametophyte development in the important crop species Brassica napus. Three cDNAs, corresponding to genes highly and specifically expressed at the tetrad and microspore stages, are presented here. The analysis of one of them, named BnM3.4, by in situ hybridization, showed that it is expressed specifically and at a high level in the rapeseed microspore. The specificity in its profile of expression is most likely transcriptionally controlled as a similar pattern of expression was also observed in Arabidopsis thaliana plants transformed by the BnM3.4 promoter fused to the reporter GUS-coding sequence. The putative BnM3.4 promoter contains three dispersed copies of a motif described previously in the promoters of several genes expressed in the male gametophyte. The BnM3.4 gene encodes a predicted novel proline-rich protein of 23.4 kDa which may interact with cytoskeletal components or have a structural role in the cell wall.