Identification of regulatory elements directing definite and specific spatiotemporal expression patterns is a prerequisite to the next generation of transgenic plants with commercial and ethical feasibility for producing plantibodies or other pharmaceutically important compounds. Here we describe the functional dissection of the barley nonspecific lipid transfer protein gene promoter, HvLTP2. The gene is specifically expressed in aleurone cells of cereals and used as an aleurone marker in maize and rice. The transcript is uniformly localised in the barley aleurone cells from around 10 DAP. Patchy expression in aleurone cells of transgenic rice has been reported and explained by silencing of transgenes. We have performed deletion analyses of the 801-bp HvLTP2 promoter to gain insight into the molecular basis of its regulation and the presence of putative regulatory elements. From the deletion studies, a 49-bp promoter region directing aleurone-specific expression was identified. Simultaneously, in vivo footprinting was carried out to identify promoter elements bound by putative regulatory proteins. Within the 49-bp fragment, the most promising candidate for a minimal cis-acting regulatory region directing aleurone specificity is the ds-sequence. Based on our results, we hypothesise that the ds-sequence directs aleurone specificity, possibly through a concerted action with elements directing general expression in the seed. Moreover, we present an overview of LTP2 elements putatively involved in directing seed, endosperm, and aleurone expression. Additionally, we report HvLTP2 expression in the embryo, not previously detected. The regulatory element(s) directing expression in embryo is located downstream of the 49-bp fragment directing aleurone specificity, thus demonstrating independent control of aleurone and embryo-localised expression. Finally, we discuss the existence of several endosperm-specific boxes and whether alternative promoter elements and combinations of them may direct aleurone expression, explaining why comparing different genes expressed in aleurone fail to identify only one required, common promoter element.