The polytene chromosome puffing pattern of Drosophila madeirensis was established and compared with those of the related species D. subobscura and D. guanche. A total of 145 loci, active in some of the 12 developmental stages analysed, were described, 38 of which were found to form the puffing pattern characteristic to this species. Taking into account the number of puffs as well as the mean puff expression, D. madeirensis shows a similar activity level to D. guanche, both species being less active than D. subobscura. The low gene activity of D. madeirensis and D. guanche was explained as a consequence of their ecological characteristics.