Sperm-mediated DNA transfer can be used to transfer exogenous DNA into the oocyte for the production of transgenic animals. In spite of controversy in the literature, sperm-mediated DNA transfer is a simple and quick technique that can be used in routine breeding programs (AI, embryo transfer and IVF). The main objective of this study was to determine the factors affecting the spontaneous uptake of exogenous DNA by bull spermatozoa. For this purpose, fresh and frozen spermatozoa (0.25 x 10(6)), from the same ejaculate from each of four bulls were co-incubated with fluorescent-labeled green fluorescent protein (GFP) and chloremphenicol acetyltransferase (CAT) plasmids at 37 degrees C for 30 min. Neither bull nor plasmid significantly affected the uptake of exogenous DNA. However, transfection efficiency was higher in frozen-thawed versus fresh spermatozoa (P<0.001). Regardless of whether transfected spermatozoa were alive or dead, all transfected spermatozoa were immotile. It can be concluded that a population of spermatozoa is present in bull semen which has the ability to uptake exogenous DNA spontaneously. There is tremendous scope to improve transfection efficiency of spermatozoa while maintaining motility; this needs to be achieved in order to more easily use this technique in transgenesis. However, live-transfected bull spermatozoa clearly can incorporate exogenous DNA and should be usable in intracytoplasmic sperm injection protocols.