The walls of grasses and related members of the Poales are characterized by the presence of the polysaccharide (1,3, 1,4)-beta-D-glucan (beta-glucan). To date, only members of the grass-specific cellulose synthase-like F (CSLF) gene family have been implicated in its synthesis. Assuming that other grass-specific CSL genes also might encode synthases for this polysaccharide, we cloned HvCSLH1, a CSLH gene from barley (Hordeum vulgare L.), and expressed an epitope-tagged version of the cDNA in Arabidopsis, a species with no CSLH genes and no beta-glucan in its walls. Transgenic Arabidopsis lines that had detectable amounts of the epitope-tagged HvCSLH1 protein accumulated beta-glucan in their walls. The presence of beta-glucan was confirmed by immunoelectron microscopy (immuno-EM) of sectioned tissues and chemical analysis of wall extracts. In the chemical analysis, characteristic tri- and tetra-saccharides were identified by high-performance anion-exchange chromatography and MALDI-TOF MS following their release from transgenic Arabidopsis walls by a specific beta-glucan hydrolase. Immuno-EM also was used to show that the epitope-tagged HvCSLH1 protein was in the endoplasmic reticulum and Golgi-associated vesicles, but not in the plasma membrane. In barley, HvCSLH1 was expressed at very low levels in leaf, floral tissues, and the developing grain. In leaf, expression was highest in xylem and interfascicular fiber cells that have walls with secondary thickenings containing beta-glucan. Thus both the CSLH and CSLF families contribute to beta-glucan synthesis in grasses and probably do so independently of each other, because there is no significant transcriptional correlation between these genes in the barley tissues surveyed.