We examined the base modification pattern of Xenopus tRNA(Sec) using microinjection into Xenopus oocytes, with particular focus on the wobble base U34 at the first position of the anticodon. We found that U34 becomes modified to mcm5U34 (5-methylcarboxymethyluridine) in the oocyte cytoplasm in a rather complex manner. When the tRNA(Sec) gene is injected into Xenopus oocyte nuclei, psi 55 and m1A58 are readily obtained, but not mcm5U34. This will appear only upon cytoplasmic injection of the gene product arising from the first nuclear injection. In contrast, tRNA(Sec) produced by in vitro transcription with T7 RNA polymerase readily acquires i6A37, psi 55, m1A58, and mcm5U34. The latter is obtained after direct nuclear or cytoplasmic injections. It has been reported by others that mcm5Um, a 2'-O-methylated derivative of mcm5U34, also exists in rat and bovine tRNA(Sec). With both the gene product and the in vitro transcript, and using the sensitive RNase T2 assay, we were unable to detect under our conditions the presence of a dinucleotide carrying mcm5Um and that would be therefore refractory to hydrolysis. We showed that the unusual mcm5U acquisition pathway does not result from impairment of nucleocytoplasmic transport. Rather, these data can be interpreted to mean that the modification is performed by a tRNA(Sec) specific enzyme, limiting in the oocyte cytoplasm.