We tested in a systematic fashion the effect of an intron on the level of luciferase expression in cultured C6/36 Aedes albopictus cells. The intron was inserted in both orientations, upstream and downstream of the luciferase coding region in two different luciferase expression vectors. The two parental luciferase expression vectors differed only in their promoters, one containing the Drosophila melanogaster actin5C promoter and the other the Autographa californica nuclear polyhedrosis virus hr5/ie1 enhancer/promoter. All resulting intron-containing constructs were tested for their ability to express luciferase in transient assays following electroporation into C6/36 cells. We found that the introns stimulate luciferase expression between twelve and sixtyfold, depending on the promoter. Enhanced expression was only seen when the intron was present in the correct orientation upstream of the luciferase ORF. When the 3' splice sites of the enhanced intron-containing constructs were mutated, the expression level dropped back to below the level of the intronless parental constructs, suggesting that the intron-dependent stimulation of luciferase expression is depending on splicing and is not due to other effects the intron may have on transcription or translation. The luciferase transcripts of all constructs were analysed by reverse transcription, PCR amplification and sequencing, and the results show a perfect correlation between efficient splicing of the intron and elevated levels of luciferase expression. Our findings have the potential to be very useful for boosting expression of foreign proteins in the widely used baculoviral or non-viral systems in insect cells.