Synthetic capped RNA transcripts produced by in vitro transcription in the presence of m(7)Gp(3)G have found a wide application in studying such processes as mRNA translation, pre-mRNA splicing, mRNA turnover, and intracellular transport of mRNA and snRNA. However, because of the presence of a 3'-OH on both m(7)Guo and Guo moieties of the cap structure, one-third to one-half of the mRNAs contain a cap incorporated in the reverse orientation. The reverse cap structures bind poorly to eIF4E, the cap binding protein, and reduce overall translational efficiency. We therefore replaced the conventional m(7)Gp(3)G cap by "anti-reverse" cap analogs (ARCAs) in which the 3'-OH of m(7)Guo moiety was substituted by 3'-deoxy or 3'-O-methyl groups, leading to m(7)3'dGp(3)G or m(2)(7,3'-O) Gp(3)G, respectively. The class of ARCAs was extended to analogs possessing an O-methyl group or deoxy group at C2' of m(7)Guo. We have also developed a series of ARCAs containing tetra- and pentaphosphates. mRNAs capped with various ARCAs were translated 1.1- to 2.6-fold more efficiently than their counterparts capped with m(7)Gp(3)G in both in vitro and in vivo systems. In a separate series, a methylene group was introduced between the alpha- and beta-, or beta- and gamma-phosphate moieties, leading to m(2)(7,3'-O)Gpp(CH2)pG and m(2)(7,3'-O)Gp(CH2)ppG. These analogs are resistant to cleavage by the decapping enzymes Dcp1/Dcp2 and DcpS, respectively. mRNA transcripts capped with m(2)(7,3'-O)Gpp(CH2)pG were more stable when introduced into cultured mammalian cells. In this chapter, we describe the synthesis of representative ARCAs and their biophysical and biochemical characterization, with emphasis on practical applications in mRNA translation.