The CEN4 sequences from chromosome 4 that impart mitotic stability to autonomously replicating (ARS) plasmids in yeast cells have been localized to a 1,755-base-pair (bp) fragment. This fragment could be cut in half to give two adjacent, nonoverlapping fragments, that each contained some mitotic stabilization sequences. One of the half-fragments worked as efficiently as the larger fragment from which it was derived, while the other half provided a much poorer degree of mitotic stabilization. Sequencing of 2,095 bp of DNA including this region revealed the presence of a centromere consensus sequence, elements I, II, and III (M. Fitzgerald-Hayes, L. Clarke, and J. Carbon, Cell 29:235-244, 1982), in the half-fragment providing high levels of mitotic stability. The poorly stabilizing half-fragment did not contain any obvious sequence homologies to other centromere sequences. Deletion analysis of the 1,755-bp fragment indicated that removal of the 14-bp element I plus 16 of the 82 bp of element II impaired mitotic stability. Removal of elements I and II eliminated the mitotic stability provided by the consensus sequence.