During mitosis in Ptk1 cells anaphase is not initiated until, on average, 23 +/- 1 min after the last monooriented chromosome acquires a bipolar attachment to the spindle--an event that may require 3 h (Rieder, C. L., A. Schultz, R. W. Cole, and G. Sluder. 1994. J. Cell Biol. 127:1301-1310). To determine the nature of this cell-cycle checkpoint signal, and its site of production, we followed PtK1 cells by video microscopy prior to and after destroying specific chromosomal regions by laser irradiation. The checkpoint was relieved, and cells entered anaphase, 17 +/- 1 min after the centromere (and both of its associated sister kinetochores) was destroyed on the last monooriented chromosome. Thus, the checkpoint mechanism monitors an inhibitor of anaphase produced in the centromere of monooriented chromosomes. Next, in the presence of one monooriented chromosome, we destroyed one kinetochore on a bioriented chromosome to create a second monooriented chromosome lacking an unattached kinetochore. Under this condition anaphase began in the presence of the experimentally created monooriented chromosome 24 +/- 1.5 min after the nonirradiated monooriented chromosome bioriented. This result reveals that the checkpoint signal is not generated by the attached kinetochore of a monooriented chromosome or throughout the centromere volume. Finally, we selectively destroyed the unattached kinetochore on the last monooriented chromosome. Under this condition cells entered anaphase 20 +/- 2.5 min after the operation, without congressing the irradiated chromosome. Correlative light microscopy/elctron microscopy of these cells in anaphase confirmed the absence of a kinetochore on the unattached chromatid. Together, our data reveal that molecules in or near the unattached kinetochore of a monooriented PtK1 chromosome inhibit the metaphase-anaphase transition.