Conserved 14-3-3 proteins have been shown to play regulatory roles in eukaryotic cells, including cell cycle control and differentiation. We were interested in the possible involvement of 14-3-3 proteins in the embryogenic process of barley (Hordeum vulgare L.). Barley microspore-derived embryo development was used as a model system. Immunolocalization of three barley 14-3-3 isoforms, 14-3-3A, 14-3-3B and 14-3-3C, was carried out using isoform-specific antibodies. In immature microspore-derived embryos, 14-3-3C was specifically expressed underneath the L<sub>1</sub> layer of the shoot apical meristem and in the scutellum. Comparative studies showed that 14-3-3C was also expressed underneath the L<sub>1</sub> layer of the shoot apical meristem and in the scutellum of immature zygotic embryos. We further demonstrated that 14-3-3C expression is restricted to L<sub>2</sub> layer-derived cells of in vitro shoot meristematic cultures. Our results reveal that 14-3-3C isoferm tissue-specific expression is closely related to defined events during differentiation processes in embryogenesis and in vitro meristematic cultures.