Establishment of embryonic stem cell (ESC) culture in pigs has not been achieved. Verification of pluripotency markers is necessary for validation of a pluripotent cell line. Not all markers observed in ESC from other species are characterized in swine embryos. The objective of this study was to characterize CD9 and α6-integrin expression in porcine blastocysts and to derive porcine ESC using Matrigel. In vitro or in vivo porcine blastocysts were submitted to total RNA extraction for RT-PCR, fixation for immunocytochemistry or immunosurgery for culture of inner cell mass. Expression of Oct-4, CD9, and α6-integrin was detected by PCR. CD9 and α6-integrin PCR products had their nucleotide sequence assessed and compared with public nucleotide database. CD9 product was identical to CD9 porcine sequences and α6-integrin product was similar to human and equine α6-integrin. Immunocytochemistry revealed Oct-4 expression in cytoplasm of the inner cell mass (ICM) and trophoblast cells. CD9 and α6-integrin were observed preferentially on trophoblast cells. No ESC colonies were obtained using co-culture on mouse embryonic fibroblasts (MEF) or on Matrigel. This study describes for the first time expression of CD9 and α6-integrin in porcine blastocysts.