Mesenchymal stem cells (MSC) have high potential in regenerative medicine such as skin substitute for a burn healing. The present study compared the characterizations of porcine MSCs (pMSC) derived from bone marrow to the same porcine adult ear and different porcine fetal skin derived cells on morphology of cell growth, alkaline phosphatase (AP) activity, cell surface (CD) markers (CD 29, 45, 90 and 105) and cell cycle by flow cytometer and protein and mRNA expression of Oct3/4, Nanog and Sox2 by immunocytochemistry and RT-PCR (Carlin et al. 2006 Reprod. Biol. Endocrinol. 4, 8) in 1–3 passage. The pMSC were isolated with Ficoll–Paque Plus (Amersham Science, USA) and skin-derived cells were separated with trypsin-EDTA solution treatment of tissue. Basal medium used for culture of pMSC and skin-derived cells was advanced-DMEM supplemented with 10% FBS and 1% penicillin–streptomycin (10 000 IU and 10 000 μg mL^–1, Gibco) and culture medium was exchanged every 3 days. Skin-derived cells were observed similar patterns of colony formation and AP expression by AP chromogen kit (BCIP/NBT) (Abcam Inc., MA, USA) to pMSC on plating culture. pMSC and skin-derived cells were found to have a stronger reaction of CD 45^– (St. Louis, MO, USA), CD 29⁺ (BD Bioscience, US), and CD 90⁺ (BD Bioscience, US). However, CD 105⁺ (abD Serotec, UK) weakly expressed in pMSC and skin-derived cells were almost negative. The population of cells at S-phase of the cycle in pMSC was significantly (P < 0.05) greater than those in adult ear and fetal-derived skin cells (30.4 ± 5.3 v. 19.1 ± 3.6 and 21.3 ± 1.7, respectively; mean ± SD). Protein of Oct3/4 (goat polyclonal IgG, Santa Cruz, CA, USA), Nanog (goat polyclonal IgG, Santa Cruz, CA, USA) and Sox2 (rabbit polyclonal IgG, Santa Cruz, CA, USA) were observed at both nucleus and cytoplasm in all cell types. However, the pattern of mRNA expression was different among cell types. Nanog and Sox2 expression was the greatest in pMSC and fetal skin cells, respectively. Oct 4 expression should read extremely low in pMSC compared with skin-derived cells, contrary to expectation. Taken together, pMSC and skin-derived cells revealed similar characterization, indicating that pMSC can expect a role of skin substitute.