Recently, studies on the 5'-ends of actin mRNAs in the nematode, Caenorhabditis elegans, have demonstrated that three of the four mature actin transcripts contain a 22-nucleotide leader sequence which is acquired by trans-splicing from a novel 100-nucleotide RNA. In the course of our studies of the ubiquitin genes in C. elegans (R. W. Graham and E. P. M. Candido, manuscript in preparation) we were led to the possibility that the major ubiquitin transcript in this organism might also contain a trans-spliced leader sequence. The 5'-noncoding region of the major ubiquitin gene (UbiA) contains a 3'-splice consensus sequence six nucleotides upstream of the initiation codon; however, sequencing of a further 1.6 kilobases upstream failed to reveal the existence of any potential 5'-splice sites in the correct relationship to known promoter elements. Furthermore, S1 and Northern blot analyses using probes complementary to upstream regions failed to detect the ubiquitin transcript. Primer extension experiments on total RNA using an oligonucleotide which hybridized to the ubiquitin transcript downstream of the 3'-splice site demonstrated the existence of a 22-nucleotide leader sequence not present in the ubiquitin gene 5'-region. Thus we show here that the 2500-nucleotide mRNA encoding a polyubiquitin precursor protein contains a 22-nucleotide leader sequence identical to that found in several C. elegans actin mRNAs and which is most likely acquired by a trans-splicing mechanism. In addition we have localized the site of transcript initiation for UbiA by S1 nuclease mapping.