The genome of the prototype foamy virus (PFV) has been introduced into an adenoviral/PFV hybrid vector and tested for stable in vitro gene transfer. Three different adenoviruses are used to encode: (i) the PFV structural genes gag and pol (Ad-GagPolDeltaPacI); (ii) the PFV structural gene env (Ad-Env); and (iii) the PFV vector genome (Ad-MD9) encoding the transgene (the enhanced green fluorescent protein (eGFP) gene). Following cotransduction by the three adenoviruses, the target cells become transient PFV vector-producing cells, resulting in the in situ release of recombinant PFV at a titre of up to 10(3) vector particles/ml, which can then infect surrounding cells, leading to stable integration of the expression cassette. Stable eGFP expression, observed for up to 60 days (11 passages) in cells transduced with all three adenoviral vectors, was shown by PCR to be the result of PFV integration. In contrast, cells transduced with only the adenovirus encoding the PFV vector genome showed a marked decrease in eGFP expression by passage 2 (16 days post-transduction) and did not contain integrated PFV vector. In short, this paper describes the production of a hybrid vector capable of high in vitro transduction and stable transgene expression using adenovirus and PFV vectors.