The effects of the Wilms' tumor suppressor protein WT1 on autonomous DNA replication under stable transfection conditions were investigated. COS ts2 monkey kidney cells, which express the simian virus 40 (SV40) replication initiator protein large tumor antigen (TAg) as a temperature-sensitive protein, were stably transfected with SV40 origin-containing plasmids expressing WT1 from the Zn2+ and Cd2+-inducible metallothionein promoter. Stable transformant clones of cells containing the integrated plasmids were isolated at the non-permissive temperature, expanded, and shifted to the permissive temperature to allow autonomous replication of the plasmid and overexpression of WT1. Expression of WT1 triggered apoptosis of the cells. Analysis of the kinetics of occurrence of cell death and accumulation of the replicated plasmid indicated that WT1 inhibited replication directly, and also indirectly by causing loss of replicated plasmid as a consequence of WT1-induced cell death.