Routine methods for stable gene transfer to cereals are not yet available. To be able to study chloroplast targeting in barley (Hordeum vulgare L.) cells, the expression of recombinant genes was assayed in barley mesophyll protoplasts after electroporation of DNA. The CaMV 35S transcript promoter was attached to a chimeric gene consisting of a pea RuBisCo small subunit transit peptide coding sequence (tp) and the gene coding for neomycin phosphotransferase II (nptII). As a control, a construction with no transit peptide coding segment was used. 48 hours after electroporation, a fraction of the protoplasts was lysed and intact chloroplasts were isolated. Protoplasts electroporated with either of the gene constructions showed strong NPTII activity. However, enzyme activity was detected in chloroplasts only when thetp-nptII gene construction was used. Protease treatment of the chloroplasts confirms that the pea RuBisCo small subunit transit peptide is targeting the NPTII polypeptide into the chloroplasts, subsequent to the synthesis of the hybrid precursor in barley cells.