Pollen germination and pollen tube elongation are important for pollination and fertilization in higher plants. To date, several pollen-specific genes have been isolated and characterized. However, there is little information about the precise spatial and temporal expression pattern of pollen-specific genes in higher plants. In our previous study, we identified 132 anther-specific genes in the model legume Lotus japonicus by using cDNA microarray analysis, though their precise expression sites in the anther tissues were not determined. In this study, by using in situ hybridization experiments, we determined the spatial and temporal expression sites of 46 anther-specific genes (ca. 35%), which were derived from two groups, cluster I-a and cluster II-a, according to flower developmental stages. In the case of the genes grouped into cluster I-a, thirteen clones were characterized. The specific hybridized signals were varied among the clones, and were observed in tapetum cells, microspores, and anther walls at the early developmental stage of anther tissues. In the case of the genes classified into cluster II-a, we used thirty three different cDNA clones encoding primary and secondary metabolism-related proteins, cell wall reconstruction-related proteins, actin reorganization-related proteins, and sugar transport-related proteins, etc., as a probe. Interestingly, all genes in these thirty three clones examined were specifically expressed in the bicellular pollen grains, though the signal intensity was varied among clones. From the data of the cluster II-a genes, the mRNAs related to pollen germination and pollen tube elongation were specifically transcribed and preserved in mature pollen grains.