Immunoglobulin class switching is mediated by recombination between switch sequences located immediately upstream of the immunoglobulin constant heavy chain genes. Targeting of recombination to particular switch sequences is associated temporally with transcription through these regions. We recently have provided evidence for inducible and stable RNA-DNA hybrid formation at switch sequences in the mouse genome that are mechanistically important for class switching in vivo. Here, we define in vitro the precise configuration of the DNA and RNA strands within this hybrid structure at the Smicro, Sgamma3 and Sgamma2b mouse switch sequences. We find that the G-rich (non-template) DNA strand of each switch sequence is hypersensitive to probes throughout much of its length, while the C-rich (template) DNA strand is essentially resistant. These results demonstrate formation of an R-loop, whereby the G-rich RNA strand forms a stable heteroduplex with its C-rich DNA strand counterpart, and the G-rich DNA strand exists primarily in a single-stranded state. We propose that the organized structure of the R-loop is essential for targeting the class switch recombination machinery to these sequences.