It is necessary to use a suitable marker gene to select embryos expressing transgene prior to transfer into recipient females especially in an efficient production of transgenic farm animals. Recently a green fluorescent protein (GFP) gene has been used as a marker gene for this purpose, because it is a self-fluo-rescent substance and does not require a substrate such as X-Gal in a lacZ gene. Using these characteristics of GFP, we transfected the GFP gene with the neomycin-resistance gene under the control of the phosphoglycerase kinase promoter into mouse embryonic stem (ES) cells, and attempted to produce a transgenic mouse derived from aggregates of GFP-posi-tive ES cells after the selection with G418 for 8 days with diploid or tetraploid embryos. There were some ES colonies expressing GFP at different levels after the selection with G418. These GFP-positive ES cells were used as a complete tight adhered clump for aggregation with diploid or tetraploid embryos and resulted in GFP expression in the inner cell mass (ICM) cells of aggregates, but no embryos expressing GFP in trophoblasts. At 9.5 days p.c., 6 out of 10 live fetuses (60%) for diploid⇔ES chimera, and all of 14 live fetuses (100%) for tetraploid⇔ES chimera, in spite of head deformity, were transgenic fetuses. At 14.5 days p.c., among 7 normal embryos obtained from 21 embryos transferred to recipients, one fetus showed GFP expression in five organs (heart, kidney, liver, gonad and intestine), and 4 fetuses strongly expressed GFP in more than one organ as seen under the fluorescent dissecting microscope. These results indicate that enough transfected ES cells selected in a short time with G418 were able to be used for the selection of transgenic embryos prior to transfer to the recipient. Moreover, by using a complete tight adhered clump of ES cells for aggregation, the clump is able to completely contribute to a part of the ICM. The GFP gene may be useful and efficient as a marker especially for farm animals which have a long term pregnancy and a small litter size.