The acid phosphatase-1 (Acph-1) gene region was sequenced in three species of Drosophila: D. subobscura, D. madeirensis and D. guanche. These three closely related species, which are included in the obscura group, form the subobscura cluster. The different functional regions of the gene were identified by similarity with the sequence of D. melanogaster. The structure of Acph-1 is conserved in the four species. Average divergence at synonymous and nonsynonymous sites between D. melanogaster and the species of the subobscura cluster is Ks = 1.1354 and Ka = 0.1743, respectively. The rather high Ka value confirms that ACPH-1 is a rapidly evolving enzyme in Drosophila, as previously suggested by immunological studies. Amino acid replacements are not randomly distributed along the gene. In fact, an excess of replacements is detected in exon I, indicating that the signal peptide encoded by this exon evolves even faster than the rest of the protein. Divergence at the Acph-1 gene region is further evidence that D. madeirensis and D. subobscura are more closely related than D. guanche is to any of them. In addition, both silent divergence in noncoding regions and synonymous divergence in the coding region indicate that the split of the D. guanche lineage is about twice as old as the split of the lineages leading to D. madeirensis and D. subobscura. These phylogenetic relationships are, however, not supported by divergence at nonsynonymous sites since the lowest Ka estimate is between D. guanche and D. subobscura.