Capped RNAs synthesized by in vitro transcription have found wide utility for studying mRNA function and metabolism and for producing proteins of interest. We characterize here a recently synthesized series of cap analogs with improved properties that contain a sulfur substitution for a nonbridging oxygen in either the alpha-, beta-, or gamma-phosphate moieties, m(2) (7,2'-O )Gppp(S)G, m(2) (7,2'-O )Gpp(S)pG, and m(2) (7,2'-O )Gp(S)ppG, respectively. The new compounds were also modified at the 2'-O position of the m(7)Guo to make them anti-reverse cap analogs (ARCAs), i.e., they are incorporated exclusively in the correct orientation during in vitro transcription. Each of the S-ARCAs exists in two diastereoisomeric forms (D1 and D2) that can be resolved by reverse-phase HPLC. A major in vivo pathway for mRNA degradation is initiated by removal of the cap by the pyrophosphatase Dcp1/Dcp2, which cleaves between the alpha- and beta-phosphates. Oligonucleotides capped with m(2) (7,2'-O )Gpp(S)pG (D2) were completely resistant to hydrolysis by recombinant human Dcp2 in vitro, whereas those capped with m(2) (7,2'-O )Gpp(S)pG (D1) and both isomers of m(2) (7,2'-O )Gppp(S)G were partially resistant. Luciferase mRNA capped with m(2) (7,2'-O )Gpp(S)pG (D2) had a t (1/2) of 257 min in cultured HC11 mammary epithelial cells compared with 86 min for m(7)Gp(3)G-capped mRNA. Luciferase mRNAs capped with m(2) (7,2'-O )Gpp(S)pG (D1) and m(2) (7,2'-O )Gpp(S)pG (D2) were translated 2.8-fold and 5.1-fold, respectively, more efficiently in HC11 cells than those capped with m(7)Gp(3)G. The greater yield of protein due to combining higher translational efficiency with longer t (1/2) of mRNA should benefit applications that utilize RNA transfection such as protein production, anti-cancer immunization, and gene therapy.