The Wnt signaling pathway is activated by mutations in the adenomatous polyposis coli (APC) or beta-catenin genes in most colon cancers, leading to the transactivation of promoters containing binding sites for the Tcf/LEF family of transcription factors. We have previously shown that it is possible to confer colon cancer specificity on autonomous parvoviruses by inserting Tcf sites into the viral P4 promoter. The mutant Tcf promoters were responsive to activation of the Wnt pathway but the viruses replicated poorly. We show here that reduction of the number of Tcf sites from four to two leads to an increase in the efficiency of replication and toxicity of the viruses in Co115 colon cancer cells, with only a small reduction in selectivity for cells with an active Wnt signaling pathway. Despite this improvement, virus production by most colon cancer cells remained low. Analysis of parental phH1 virus infection of SW480 colon cancer cells showed that the nonstructural and capsid proteins were expressed, but single stranded DNA and progeny virus were not produced. This defect reflects the dependence of autonomous parvoviruses on host functions for many steps in their replication cycle and represents a major limitation to the use of selectively replicating parvoviruses for colon cancer therapy.