A gene transfer method to ectopically express genes during chicken limb pattern formation using replication defective retroviral vectors has been established. Spherical non-proliferating (mitomycin C treated) aggregates of clonal retrovirus producing cells were grafted directly into developing chicken wing buds. The cell aggregates had to be placed in direct contact with the highly proliferative cells of the wing bud to promote efficient in vivo infection of embryonic cells by the released retroviral particles. The majority of grafts resulted in widespread expression of a reporter gene (encoding bacterial beta-galactosidase) during limb pattern formation and early limb bud outgrowth without affecting morphogenesis. This method provides a novel approach to study the effects of ectopic gene expression on limb pattern formation. Possible future applications to study other developmental processes are discussed.