The Signal Peptide Peptidases (SPP) are members of the Intramembrane Cleaving Proteases, which are involved in an array of protein-processing and intracellular signaling events in animals. Arabidopsis (Arabidopsis thaliana) has six genes encoding SPP-like proteins, the physiological functions of which are unknown. As a first step in defining the roles of the SPPs in plants, we examined the distribution and activities of Arabidopsis SPP (AtSPP; accession no. At2g03120), the SPP-like gene with the highest degree of similarity to human SPP. The protease is expressed at low levels throughout the plant, with the highest levels in emerging leaves, roots, and floral tissues. Homozygous plants carrying a T-DNA insertion mutation in AtSPP, spp-2, could not be recovered, and transmission of the mutant allele through pollen was reduced to less than 2% in reciprocal cross experiments. Although viable, pollen from spp-2 heterozygous plants exhibited a 50% reduction in germination rate and a disruption in male germ unit organization. These data demonstrate that AtSPP is required for male gametophyte development and pollen maturation in Arabidopsis.