Arabidopsis UDP-sugar pyrophosphorylase (AtUSP) is a broad substrate enzyme that synthesizes nucleotide sugars. The products of the AtUSP reaction can act as precursors for the synthesis of glycolipids, glycoproteins, and cell wall components including pectin and hemicellulose. AtUSP has no close homologs in Arabidopsis and its biological function has not been clearly defined. We identified two T-DNA insertional mutant lines for AtUSP, usp-1 and usp-2. No homozygous individuals were identified and progeny from plants heterozygous for usp-1 or usp-2 showed a 1:1 segregation ratio under selection. Despite decreased levels of both AtUSP transcript and USP activity (UDP-GlcA-->GlcA-1-P), heterozygous plants were indistinguishable from wild type at all stages of development. Reciprocal test crosses indicated the source of the segregation distortion was lack of transmission through the male gametophyte. Analysis of pollen tetrads from usp-1 in the quartet background revealed a 2:2 ratio of normal:collapsed pollen grains. The collapsed pollen grains were not viable as determined by Alexander's viability and DAPI staining, and pollen germination tests. The pollen phenotype of usp-1 was complemented by transformation of usp-1 with the AtUSP cDNA sequence. Surface and ultrastructural analyses of pollen from wild-type and usp mutants demonstrated that the mutation had no apparent effect on the outer wall (exine) but prevented the synthesis of the pectocellulosic inner wall (intine). Evidence presented here shows that AtUSP has a critical role in pollen development.