The N(7)-methyl guanosine cap located on the 5'-terminus of mRNAs is important for a number of biochemical processes. A new dinucleoside triphosphate cap analog was synthesized with methyl groups on the N(7) of both guanine moieties, as well as the 3'-OH of one of the ribose moieties [see text]. The function of this trimethylated cap analog was compared with those of three other, less-methylated cap analogs: one omitting the ribose methylation (m(7)G[5']ppp[5']m(7)G), one omitting the N(7) methylation linked to the unmodified ribose [see text], and the standard cap analog, m(7)G[5']ppp[5']G. These cap modifications were assayed with respect to their effects on capping efficiency, yield of RNAs during in vitro transcription, and the translational activity of these RNAs upon transfection into HeLa cells. The translational activity was monitored by measuring the luciferase activity of a luciferase-fusion protein produced from the in vitro synthesized RNAs. The RNA capped with the trimethylated analog [see text] was translated the most efficiently, with approximately 2.6-fold more activity than the conventional cap (m(7)G[5']ppp[5']G). The other two variants were also more efficient, generating, approximately 2.2 times (for the [see text] analog) and, approximately 1.6 times (for the m(7)G[5']ppp[5']m(7)G analog) more luciferase function than the conventional cap.