This study describes a protocol for Agrobacterium-mediated transformation of barley mature embryos. Cultured embryos were co-cultivated by EHA 105 strain carrying pCambia2301 binary vector and assayed for transient GUS expression. After 3 days of transformation, GUS expressing spots have been observed over 20.3% of total embryos. Among the tested several pre-treatments, infiltration in a vacuum chamber (400 mm Hg for 20 min) and infection by AGL-1 strain instead of EHA 105 increased the numbers of embryos with GUS staining significantly (p<0.05). PCR and Southern analyses showed that transformation occured in some of the T0 plants which were also tolerant to selective conditions