The phenomenon of pre-mRNA splicing in eukaryotes has been mostly studied in mammalian and yeast systems. The splicing machinery in plants is thought to be largely conserved relative to animal and fungal organisms. This thesis encompasses systematic studies of splicing-related genes and alternative splicing (AS) in plants. A total of 74 snRNA genes and 395 genes encoding splicing related proteins were identified in Arabidopsis, including the previously elusive U4atac snRNA gene. About 50% of the splicing related genes are duplicated in plants. The duplication ratios for splicing regulators are even higher, indicating that the splicing mechanism is generally conserved among plants, but that the regulation of splicing may be more variable and flexible.;Over 30% of the splicing related genes can be alternatively spliced. Overall, both Arabidopsis and rice have about 22% of the expressed genes being alternatively spliced, and both have about 55% AS events to be intron retention (IntronR). The consistent high frequency of IntronR suggests prevalence of splice site recognition by intron definition in plants. 40% of Arabidopsis AS genes are also alternatively spliced in rice, with some examples strongly suggesting a role of the AS event as an evolutionary conserved mechanism of post-transcriptional regulation.;U2AF is an essential splicing factor in animals. The two copies of Arabidopsis U2AF1 (AUSa and AUSb) were experimentally characterized as a case study. AUSa expressed at a higher level than AUSb in most tissues. Altered expression levels of AUSa or AUSb cause pleiotropic phenotypes and splicing pattern changes for some pre-mRNA, indicating the importance of AUSa/b for correct splice site recognition. A novel C-terminal domain (SERE) is highly conserved in all seed plant U2AF1 homologs, suggesting its important function specific to higher plants.;All together, similarities as well as differences were revealed between the splicing mechanisms in plants and mammalians, demonstrating that organisms have evolved special mechanisms to ensure the efficient and accurate splicing in different environments. Two databases (Arabidopsis Splicing Related Genes (ASRG), http://www.plantgdb.org/SRGD/ASRG/, and Alternative Splicing in Plants (ASIP), http://www.plantgdb.org/ASIP/) were constructed for the community to use and will facilitate studies of plant splicing mechanisms.