We investigated the effect of extracellular matrix protein on in vitro attachment and outgrowth of bovine hatched blastocysts. In vitro produced bovine hatched blastocysts were cultured on a fibronectin- or laminin-coated Petri dishes. Hatched blastocysts adhered and outgrew on the fibronectin-coated dish whereas no attachment was observed on the laminin-coated dish. The attachment and outgrowth on fibronectin were significantly inhibited in the presence of synthetic peptides containing the Arg-Gly-Asp (RGD) sequence, which interacts with the fibronectin receptor (integrin alpha5beta1), but were not inhibited by the control peptides containing the Arg-Gly-Glu (RGE) sequence. Addition of anti-fibronectin receptor (integrin alpha5beta1) antibody to the culture medium also inhibited the attachment and outgrowth on fibronectin-coated Petri dishes. Subsequently we examined mRNA expression and protein expression of alpha5 and beta1 integrin subunit in the hatched blastocyst by reverse transcription-polymerase chain reaction (RT-PCR) and immunostaining, respectively. Expression of both mRNA and protein were detected in blastocysts. These results indicate that trophectoderm cells of bovine hatched blastocysts have already acquired the ability to adhere and outgrow on fibronectin in vitro by an integrin- mediated manner.