It is desirable to produce transgenic plants which have optimized and stable levels of transgene expression. Low levels of transgene expression may lead to an insufficient quantity of transgenic protein being produced for a particular purpose. This report demonstrates a means of enhancing transgene expression in barley beyond that conferred by the Ubi1 promoter, via the inclusion of an intron at a specific position within the transgene coding sequence. We independently cloned two different introns (RpoT-i4 from maize and UBQ10-i1 from Arabidopsis) into the same position within the firefly luciferase (luc) coding sequence. The constructs produced were transformed into barley (Hordeum vulgare) via Agrobacterium-mediated transformation, and the resulting transformant populations (of between 119 and 123 independent plants for each construct) were assayed for luciferase activity. Both introns significantly increased luciferase activity, and a quantitative reverse-transcription polymerase chain reaction assay revealed that the introns increased the accumulation of luciferase mRNA transcripts. The enhanced transgene expression levels were maintained in the T(1) and T(2) progenies. These findings show that intron-mediated enhancement is a valuable additional tool for achieving high and stable levels of transgene expression in crop plants.