AAA+ proteins form a large superfamily of P-loop ATPases involved in the energy-dependent unfolding and disaggregation of macromolecules. In a clustering study aimed at defining the AAA proteins within this superfamily, we generated a map of AAA+ proteins based on sequence similarity, which suggested higher-order groups. A classification based primarily on morphological characteristics, which was proposed at the same time, differed from the cluster map in several aspects, such as the position of RuvB-like helicases and the inclusion of divergent clades, such as viral SF3 helicases and plant disease resistance proteins (RFL1). Here, we establish the presence of an alpha-helical domain C-terminal to the ATPase domain (the C-domain) as characteristic for AAA+ proteins and re-evaluate all clades proposed to belong to this superfamily, based on this characteristic. We find that RFL1 and its homologs (APAF-1, CED-4, MAT, and AfsR) are AAA+ proteins and SF3 helicases are not. We also present a new and more comprehensive cluster map, which assigns a central position to RuvB and clarifies the relationships between the clades of the AAA+ superfamily. (c) 2006 Elsevier Inc. All rights reserved.