We successfully produced second-generation cloned cats by somatic cell nuclear transfer (SCNT) using skin cells from a cloned cat. Skin cells from an odd-eyed, all-white male cat (G0 donor cat) were used to generate a cloned cat (G1 cloned cat). At 6 months of age, skin cells from the G1 cloned cat were used for SCNT to produce second-generation cloned cats. We compared the in vitro and in vivo development of SCNT embryos that were derived from the G0 donor and G1 cloned donor cat's skin fibroblasts. The nuclei from the G0 donor and G1 cloned donor cat's skin fibroblasts fused with enucleated oocytes with equal rates of fusion (60.7% vs. 58.8%, respectively) and cleavage (66.3% vs. 63.4%). The 2-4-cell SCNT embryos were then transferred into recipients. One of the five recipients of G0 donor derived NT embryos (20%) delivered one live male cloned kitten, whereas 4 of 15 recipients of the G1 cloned donor cat derived NT embryos (26%) delivered a total of seven male second-generation cloned kittens (four live kittens from one surrogate, plus two stillborn kittens, and one live kitten that died 2 d after birth from three other surrogate mothers). The four second-generation cloned kittens from the same surrogate all had a white coat color; three of the four second-generation cloned kittens had two blue eyes, and one of the second-generation cloned kittens had an odd-eye color. Despite low cloning efficiency, cloned cats can be used as donor cats to produce second-generation cloned cats. #