Design, synthesis, and biological evaluation of 2'-fluoro-substituted cap analogs, i.e., m(7,2'F)G[5']ppp[5']G and m(7,2'F)G[5']ppp[5']m(7)G are described. Structures were confirmed by (1)H, (31)P, (19)F NMR and MS data. The effects of the 2'-fluoro-substituted moiety from the normal and N(7) double methylated mCAP were evaluated with respect to their capping efficiency, in vitro T7 RNA polymerase transcription efficiency, and translation activity using cultured HeLa cells. Luciferase fusion protein production was monitored by measuring the luciferase activity. mRNA poly(A) capped with 2'-fluoro-substituted cap analogs, (m(7,2'F)G[5']ppp[5']G) and (m(7,2'F)G[5']ppp[5']m(7)G), were translated approximately 2.4- and 2.5-fold more efficiently, respectively, than mRNA capped with conventional m(7)G[5']ppp[5']G.