As the biochemical detection of bovine papillomavirus type 4 E5 is problematic, a fusion form of E5 and the green fluorescent protein (GFP-E5) was constructed and its characteristics were examined. GFP-E5 was detected in cells by autofluorescence and immunoblotting. Like wild-type (wt) E5, GFP-E5 localized in the endomembranes and permitted anchorage-independent (AI) growth. However, unlike wt E5, cells expressing GFP-E5 became quiescent in low serum and failed to sustain expression of cyclins D1 and to inactivate retinoblastoma protein (pRb). The normal anchorage requirement for cyclin D1 and cyclin A expression was abolished in cells expressing wt E5 or GFP-E5, residual extracellular signal-regulated kinase (ERK 1/2) activity was not required to sustain cyclin D1 and cyclin A expression in suspension and deregulation of cyclin A-cyclin-dependent kinase (CDK) activity was sufficient to account for AI growth of cells expressing E5. Constitutive upregulation of the CDK inhibitor p27 KIP1 , characteristic of cells expressing wt E5, was not observed in those expressing GFP-E5; therefore, p27 KIP1 deregulation is not required for E5-mediated AI growth.