Initiation events occur throughout the subtelomere in the human 5p segment in three cell lines, and replication forks progress through the segment in both directions. SMARD analysis of three different cell lines indicated that the 5p telomere is replicated primarily by forks progressing from the subtelomere to the telomere. Alignments of replicated molecules fully labeled with both IdU (red) and CldU (green) are shown. A map of the 5p locus is depicted above each alignment, with the positions of the FISH probes (blue bars below) used for identifying and orienting the molecules indicated. Vertical orange lines indicate the positions of the ends of the subtelomeric FISH signals used to align the molecules. The boundary between the subtelomere and telomere is delineated by a vertical blue line. Yellow arrows mark sites of transition from IdU incorporation to CldU incorporation and indicate the direction of fork progression at the moment of transition during the replication of the molecule. Replication profiles, histograms of the percentage of molecules containing IdU per 5-kb interval along the segment, are shown below each alignment. Initiation events (red tracts surrounded by green) occur at multiple locations. The origins appear to be clustered around a 40–90-kb region centered 65–115-kb from the telomere, seen as a peak in the replication profiles. Aside from a leftward broadening of the initiation zone in IMR-90 cells, there was not much variation in the basic features of the replication program of the 5p telomere in the two ES cell lines (H1 and H9) and the primary fibroblasts (IMR-90).