Since the first successful somatic cell nuclear transfer (SCNT) experiments were carried out, a number of domestic and agriculture species have been cloned using donor cells derived from different sources and origin. However, differences in nuclear transfer (NT) efficiency both in vitro and in vivo have been generally observed. These differences may be accentuated when transgenic cell lines are used as nuclear donors in an attempt to generate transgenic cloned offspring. The present study examined the suitability of cell lines derived from 3 different fetal sources and the effects of genetic manipulation of donor fetal fibroblasts with a red fluorescent plasmid, on the in vitro developmental potential and quality of NT derived bovine embryos. We observed no differences in the cleavage rate of cloned embryos generated with any of the cell lines evaluated. However, the blastocyst rate was significantly affected when cell lines were derived from the 3 different fetal sources (21, 18 and 11%, respectively) or from 2 transgenic clonal cell lines that had originated from the same primary fetal cell (18 and 10%, respectively). Despite this difference, quality of embryos as measured by the total number of cells and by assessing some morphology aspects of their appearance was not different. Together these results indicate that fetal fibroblast cell lines derived from different fetal sources and transgenic clonal cell lines that had originated from the same fetus results in different in vitro developmental potential when used as donors for NT experiments. Further studies, including evaluation of pregnancy rates, development to term, and epigenetic modifications of these cell lines will be necessary to better understand the differences observed in NT efficiency.