HeLa cells expressing a kinetochore-specific protein were synchronized and released into S phase. At time intervals of 1,3,4,6,and 8 hr, cells were pulsed with BrdU, fixed and processed to label contromeres (red) and replicating DNA (green). Nuclear DNA was stained with DAPI (blue). Z-series image stacks were obtained using widefield microscopy, and the out-of-focus signal removed by constrained iterative deconvolution. Each column shows a single nucleus representative of the S-phase stages I - V. The top row (a-e) shows a merge of the red, green and blue signals, and the center row (f-j), the red and green signals only. The red and green signals were processed to reveal centromere-associated DNA replication which peaks in the latter half of S.