Nucleotide variation at the nuclear ribosomal protein 49 (rp49) gene region has been analysed by fine restriction mapping in a sample of 47 lines from a population from Madeira. Five restriction-site (out of 37 sites scored) and 3 length polymorphisms have been detected, resulting in 14 different haplotypes. This population shows less variation than both continental and Canary Island populations. The population from Madeira shows some differentiation from mainland populations, which does not favor the idea of extensive migration between the continent and Madeira. Chromosomal and restriction-map variation of the rp49 region in D. subobscura populations, together with data on sequence comparison of this nuclear region in D. guanche and D. madeirensis clearly indicate that the Canary Islands underwent at least two colonization events from the nearby continent. Although the data for Madeira are compatible with a single colonization event by a continental sample polymorphic for gene arrangements O3 and O3 + 4, an alternative scenario with at least two colonization events seems more likely.