Phylogenetic affinities among taxa associated with the Nearctic component of the Anopheles maculipennis species group (subgenus Anopheles) were inferred from sequence divergence in the D2 variable region of 28S ribosomal RNA. The base composition of this region had a marked GC bias which ranged from 59.9% in Anopheles walkeri to 65.1% in Anopheles punctipennis E. Although over two-thirds of the base positions in the D2 region were double-stranded (stem), substitution frequencies at single-stranded (loop) positions (0.068 over all taxa) were 2.7 times greater than at stem positions (0.025). Most mutations were point mutations and were most frequent at loop positions. In the shortest trees generated by both parsimony and distance methods, the four American species traditionally identified with the maculipennis complex (Anopheles aztecus, Anopheles earlei, Anopheles freeborni, and Anopheles occidentalis) were monophyletic with A. punctipennis E and W as sister taxa. The latter two correspond to genetically distinct forms from the eastern United States and California, respectively. The sibling species of the Anopheles quadrimaculatus complex formed a distinct clade, and A. quadrimaculatus D, with six autapomorphies, was the most divergent of these taxa. Sequence divergence between A. walkeri and the other taxa included in the study was of such magnitude as to suggest only a distant affinity to these species.