Transgenic animals have been successfully produced by mass gene transfer techniques such as sperm-mediated gene transfer (SMGT). The aim of this work was to demonstrate transgene transmission by SMGT in chickens using dimethylsulfoxide (DMSO) or N,N-dimethylacetamide (DMAc) as transfectants after seminal plasma removal to prevent DNase activity. Sperm samples were prepared by repetitive washes, and after each wash sperm motility, seminal plasma proteins, exogenous DNA integrity and its uptake by spermatozoa were evaluated. Laying hens were inseminated using spermatozoa transfected with pEGFP-N1 vector in the presence of DMSO or DMAc. Transgene transmission in newborn chicks was evaluated by in vivo enhanced green fluorescent protein (EGFP) expression, RT-PCR and PCR analysis. DNA internalization was limited to sperm samples washed twice. The presence of DMSO or DMAc during transfection had no effect on fertilization or hatching rates. PCR analysis detected the presence of EGFP DNA in 38% of newborn chicks from the DMSO group and 19% from the DMAc group. EGFP mRNA was detected in 21% of newborn chicks from the DMSO group, as against 8.5% from the DMAc group. However, in vivo expression of EGFP was only observed in a single animal from the DMSO group. Our data revealed that the plasmid DNA-DMSO combination coupled with sperm washes can be an efficient method for transfection in chickens.