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Abstract In previous work, we characterized several cDNA and genomic clones of genes expressed in the pollen of alfalfa (Medicago sativa L.), also known as lucerne. In this study, we have extended that work and focused on expression at the protein level of one of those genes, PO2, a low-copy gene of unknown function, but with similarities to arabinogalactan proteins. Polyclonal antibodies, specific to the PO2 protein expressed in Escherichia coli, were raised in chickens, extracted from the eggs, and used in Western blots and immunolocalization studies to identify the location of the PO2 protein in sections of mature alfalfa pollen grains and pollen tubes. Our Western blot and immunolocalization data suggest that the PO2 protein is secreted, occurs largely in the intine, and is not detectable in the pollen tube; moreover, it is not detectable on the surface of the pollen grain, nor does it diffuse out of the pollen grain into an aqueous medium.