Abstract In the previous paper , we reported a specific karyotypic change in the cell which is a clonal line derived from HeLa-S3 cell and is persistently infected with hemadsorption type 2 virus (S3/HA2). The modal number of the chromosome in S3/HA2 was 68, hypotriploidy as was in the uninfected control S3 cell. The karyotypic change, however, occurred in virtually all of the S3/HA2 cells and was characterized by the following features: each one of the X-chromosomes and the G-group chromosomes was missing and two chromosomes were added to the F-group chromosomes. The present autoradiographic study made comparatively on both the uninfected control S3 and S3/HA2 cells not only confirmed the previous findings but also revealed the following facts: In control S3 cells, the X-chromosome consisted of two extremely late-replicating chromosomes and one relatively early-replicating chromosome. A member of the G-21 chromosome replicated later than that of the G-22 and all of the F-group chromosomes were of the early-replicating chromosome. In contrast, the metaphases of the S3/HA2 cells exhibited distinctly different labeling pattern from that of the control S3 cells. A late-replicating chromosome was added to each of the F-19 and F-20 chromosomes, all of which were of the early-replicating chromosome in the control S3 cell. In addition, one of the extremely late-replicating X-chromosomes and one of the late-replicating G-21 chromosomes were missing. On the basis of these observations, a possible mechanism of the karyotypic change involved in the S3/HA2 cell was discussed.