The study of large animal embryonic stem cells (ESCs) in vitro has implications for the understanding of lineage differentiation and transgenesis. The first step for ESC derivation is the attachment of the embryo to a substrate on which they can form outgrowths. However, the culture conditions for large animal embryo attachment and ESC derivation have not been studied extensively. Defining culture conditions for embryo attachment such as culture medium and substrate is an important first step for derivation of inner cell massderived stem cells. The aim of this study was to compare different types of culture media and substrates for their ability to support attachment of in vitro produced bovine embryos in culture. Bovine embryos were produced in vivo following established protocols. Blastocysts formed on day 8 after fertilization were transferred to 12-well culture plates containing different types of culture media (Dulbecco's Modified Eagle Medium, DMEM or Medium 199, M199) and substrates [bovine fetal fibroblasts, goat fetal fibroblasts, mouse embryonic fibroblasts (STO) or non-cellular substrates (gelatin, laminin, fibronectin)]. Percentage of attached embryos and number of days since fertilization required for attachment were recorded. Bovine blastocysts preferrably attached to feeder cells rather than noncellular substrates and there was an interaction of feeder cell type and culture medium used. Therefore, the choice of both feeder cell type and culture medium has to be considered when optimizing conditions to derive cell lines from bovine embryos.