B lymphocytes can alter selectively their immunoglobulin (Ig) isotype expressed by deletional rearrangement of the first active immunoglobulin heavy-chain (IgH) constant region (C mu) gene with one of six other constant region genes. Recombination breakpoints occur within highly repetitive "switch" (S) regions located upstream of each IgH constant region gene except C delta. Analysis of rearranged switch DNA junctions has not detected a consensus sequence, although the predominance of two pentamer motifs (TGGGG and TGAGC) at or near these breakpoints and throughout all murine S region sequences has led to their advocacy as the S recombination signals. In this paper, we describe the characterization and partial purification of a lymphoid-specific endo-nuclease activity which cleaves preferentially murine S region DNA. Enzyme activity selectively produced single- and double-stranded breaks at TGAGC and TGGG motifs within murine S mu and S alpha DNA. Rare cryptic cleavage sites were detected also within non-switch sequences, although cleavage intensities at these sites were reduced greatly, relative to consensus S region cleavages. Analogous activity was found in murine tissue extracts, although among the tissues assayed only spleen and thymus contained detectable activity. Subsequent biochemical characterization of this activity demonstrated that the responsible enzyme (Endo-SR) represented a previously unreported tissue-specific mammalian endonuclease. Endo-SR-specific activity could be enhanced by addition of Mg2+ or Ca2+ and inhibited by addition of Zn2+. Maximal specific activity was detected at pH 5.5 and sharply declined within +/- 0.5 pH units. In view of this enzyme's sequence- and tissue-specificity, we propose that Endo-SR is a strong candidate for an endonuclease activity associated with the switch recombination process.