Poly(A) polymerase is a key enzyme in the machinery that mediates mRNA 3' end formation in eukaryotes. In plants, poly(A) polymerases are encoded by modest gene families. To better understand this multiplicity of genes, poly(A) polymerase-encoding genes from several other plants, as well as from Selaginella, Physcomitrella, and Chlamydomonas, were studied.Using bioinformatics tools, poly(A) polymerase-encoding genes were identified in the genomes of eight species in the plant lineage. Whereas Chlamydomonas reinhardtii was found to possess a single poly(A) polymerase gene, other species possessed between two and six possible poly(A) polymerase genes. With the exception of four intron-lacking genes, all of the plant poly(A) polymerase genes (but not the C. reinhardtii gene) possessed almost identical intron positions within the poly(A) polymerase coding sequences, suggesting that all plant poly(A) polymerase genes derive from a single ancestral gene. The four Arabidopsis poly(A) polymerase genes were found to be essential, based on genetic analysis of T-DNA insertion mutants. GFP fusion proteins containing three of the four Arabidopsis poly(A) polymerases localized to the nucleus, while one such fusion protein was localized in the cytoplasm. The fact that this latter protein is largely pollen-specific suggests that it has important roles in male gametogenesis.Our results indicate that poly(A) polymerase genes have expanded from a single ancestral gene by a series of duplication events during the evolution of higher plants, and that individual members have undergone sorts of functional specialization so as to render them essential for plant growth and development. Perhaps the most interesting of the plant poly(A) polymerases is a novel cytoplasmic poly(A) polymerase that is expressed in pollen in Arabidopsis; this is reminiscent of spermatocyte-specific cytoplasmic poly(A) polymerases in mammals.