Calcium plays an essential role during pollen development and pollen tube growth, and several Ca(2+)-binding proteins are expressed in anthers. We have previously reported that Brassica pollen allergens encoded by Bra r 1 and Bra r 2 show sequence similarities to Ca(2+)-binding proteins [Toriyama et al. (1995) Plant Mol. Biol. 29: 1157]. Herein, we report that both genes are expressed in the diploid tapetum and haploid microspores, as detected by in situ RNA hybridization. Immunoblot analysis revealed that Bra r 1 and Bra r 2 were accumulated in anthers during pollen development. When pollen grains were suspended in an aqueous solution, both proteins were mainly detected in the pollen extracellular fraction, indicating that Bra r 1 and Bra r 2 are released from the pollen upon hydration. Localization of Bra r 1 was further investigated in sections of anthers and pollen tubes. Bra r 1 was detected in the tapetum, microspores and pollen grains. In longitudinal sections of cross-pollinated pistils. Bra r 1 was detected throughout pollen tubes elongating in transmitting-tissue. These findings suggest that Bra r 1 may be involved in pollen-pistil interaction and pollen tube growth.