Eukaryotic precursor tRNAs undergo extensive processing prior to nuclear export. The first of multiple factors to interact with pre-tRNAs and other nascent transcripts is the La protein. Using suppressor and wild-type tRNAs, we demonstrate that the normal distribution of cellular end-processed and spliced tRNA species is disordered by La proteins that lack a conserved nuclear retention element. Fission yeast or human La mutants that lack this element enter nuclei and stabilize nascent pre-tRNA but are aberrantly exported and fail to support normal tRNA processing. Instead, anomalous 5' and 3' end-containing, spliced tRNAs accumulate, complexed with the mutant La protein. Thus, appropriate nuclear trafficking by La affects the normal order of pre-tRNA processing.