In mammals, cloned individuals can be produced from somatic cells. The combined use of gene targeting in embryonic stem cells and cloning contributes to the investigation of gene function in mammals. However, one of the major limitations to cloning is the low viability of cloned embryos, leading typically to high rates of pre- and postnatal death. The present study investigated whether cloning efficiency is influenced by the procedural differences involved in using transfected embryonic stem cells arrested at M phase for cloning by both single and serial transfer. In contrast to a previous study, in which fibroblasts were used, in the present study using embryonic stem cells there was no difference in the rate of production of cloned pups after the use of a single or serial nuclear transfer, although the proportion of blastocysts (70% versus 51%) was significantly higher (P < 0.001) after serial nuclear transfer. After embryo transfer of 445 blastocysts, 218 (49%) implanted and 27 (6% of blastocysts transferred) live pups were born. Of these 27 pups, 23 developed to adults of apparently normal fertility. Of these adults, 39% (n = 9) were derived from targeted embryonic stem cells, which is similar to the proportion of targeted embryonic stem cells in the population used for cloning. This study showed that cloning with embryonic stem cells is a viable procedure resulting in the production of transgenic cloned adults.