EXON sequences present on separate RNA molecules can be joined by trans-splicing in trypanosomatids, Euglena, and in the nematode and trematode worms^1–3. Trans-splicing involves an interaction between a 5′ splice site present in a spliced leader RNA and a 3′ splice site located near the 5′ end of pre-messenger RNAs. In vitro trans-splicing of artificial mammalian pre-mRNAs has been reported, but the efficiency of splicing appears to depend on sequence complementarity between the two substrates^4–7. There has been speculation that some natural pre-mRNAs can be trans-spliced in mammalian cells in vivo^8,9, but alternative interpretations have not been ruled out. Here we show that spliced leader RNAs can be accurately trans-spliced in mammalian cells in vivo and in vitro. Both nematode and mammalian 3′ splice sites can function as acceptors for trans-splicing in vivo. These results reveal functional conservation in the splicing machinery between lower eukaryotes and mammals, and they directly demonstrate the potential for trans-splicing in mammalian cells.