The male gametophyte of Arabidopsis is a three-celled pollen grain that is thought to contain almost all the mRNAs needed for germination and rapid pollen tube growth. We generated a reference map of the Arabidopsis mature pollen proteome by using multiple protein extraction techniques followed by 2-DE and ESI-MS/MS. We identified 135 distinct proteins from a total of 179 protein spots. We found that half of the identified proteins are involved in metabolism (20%), energy generation (17%), or cell structure (12%); these percentages are similar to those determined for the pollen transcriptome and this similarity is consistent with the idea that in addition to the mRNAs, the mature pollen grain contains proteins necessary for germination and rapid pollen tube growth. We identified ten proteins of unknown function, three of which are flower- or pollen-specific, and we identified nine proteins whose RNAs were absent from the transcriptome, seven of which are involved in metabolism, energy generation, or cell wall structure. Our work complements and extends recent analyses of the pollen transcriptome.