The Acph-1 gene region was sequenced in 51 lines of Drosophila subobscura. Lines differ in their chromosomal arrangement for segment I of the O chromosome (O(st) and O(3+4)) and in the Acph-1 electrophoretic allele (Acph-1(100), Acph-1(054), and Acph-1(>100)). The ACPH-1 protein exhibits much more variation than previously detected by electrophoresis. The amino acid replacements responsible for the Acph-1(054) and Acph-1(>100) electrophoretic variants are different within O(st) and within O(3+4), which invalidates all previous studies on linkage disequilibrium between chromosomal and allozyme polymorphisms at this locus. The Acph-1(>100) allele within O(3+4) has a recent origin, while both Acph-1(054) alleles are rather old. Levels of nucleotide variation are higher within the O(3+4) than within the O(st) arrangement except for nonsynonymous sites. The McDonald and Kreitman test shows a significant excess of nonsynonymous polymorphisms within O(st) when D. guanche is used as the outgroup. According to the nearly neutral model of molecular evolution, this excess is consistent with a smaller effective size of O(st) relative to O(3+4) arrangements. A smaller population size, a lower recombination, and a more recent bottleneck might be contributing to the smaller effective size of O(st).