ARTICLE ABSTRACTReplicative senescence limits the number of times primary cells can divide and is therefore regarded as a potential checkpoint for cancer progression. The majority of studies examining changes of gene expression upon senescence have been made with stationary senescent cells. We wanted to study the transition from normal growth to senescence in detail and identify early regulators of senescence by analyzing early changes in global gene expression, using Affymetrix microarrays. For this purpose, we used a murine epithelial senescence model, where senescence is abrogated by SV40 large T antigen and can be induced by using a temperature-sensitive form of SV40 large T antigen (SV40ts58). Comparisons were made to wild-type SV40 large T antigen-expressing cells and to cells expressing SV40ts58 large T antigen grown to confluence. After removal of genes that are similarly regulated in wild-type and temperature-sensitive SV40 large T antigen-expressing cells, 60% of the remaining genes were shared between cells arrested by inactivation of SV40 T antigen and by confluence. We identified 125 up-regulated and 39 down-regulated candidate genes/expressed sequence tags that are regulated upon SV40 T antigen inactivation and not during heat shock or confluence and classified these based on their kinetic profiles. Our study identified genes that fall into different functional clusters, such as transforming growth factor-β-related genes and transcription factors, and included genes not identified previously as senescence associated. The genes are candidates as early regulators of the senescence checkpoint and may be potential molecular targets for novel anticancer drugs.