Thousands of programmed genomic deletion events occur during macronuclear development in Tetrahymena thermophila. Two of the deleted segments, called M and R, have been particularly well-characterized. Using ligation-mediated PCR, we have detected DNA strand breaks that correlate temporally and structurally with the deletion events in the M and R regions. The ends appear at positions that correspond precisely to boundaries of deleted sequences, as defined by observed chromosomal junctions found after deletion is complete. They occur exclusively during the known DNA rearrangement period in macronuclear development. The breaks are staggered by 4 bp in the complementary strands. Several alternative breaks were found at the end of one deleted region, consistent with multiple alternative chromosomal junctions detected previously. The free 5' ends generated at the breaks are phosphorylated. A purine residue always occurs at the free 3' ends, with an adenosine appearing in 11 of 12 cases. Patterns found in the detected break sites suggest rules that define the ends of the deleted segments within a transposon-like deletion mechanism.