Ig class switch recombination (CSR) in activated B cells is preceded by the generation of "switch" transcripts from the heavy chain constant region (CH) genes targeted for rearrangement. Switch transcripts include a sterile "I" exon spliced onto the first CH exon. Targeted mutations disrupting the expression or splicing of I exons severely hamper CSR to all tested CH loci, except mu. However, all mu switch transcript mutations tested so far have left the I mu exon splice donor site intact. To test the possibility that the residual CSR activity in I mu mutants could be due to splicing of a truncated I mu exon, we generated new mutants specifically lacking the I mu splice donor site. Surprisingly, normal CSR was observed in the I mu splice donor mutants even in the absence of detectable spliced I mu transcripts. In a search for potential alternative sources of switch-like transcripts in the mu locus, we identified two novel exons which map just upstream of the Smu region and splice onto the C mu 1 exon. Their expression is detectable from early B cell developmental stages, and, at least in hybridomas, it does not require the Emu enhancer. These studies highlight a unique structure for the mu locus I exon region, with multiple nested switch transcript-like exons mapping upstream of Smu. We propose that all of these transcripts directly contribute to mu class switching activity.