In addition to conventional antibodies (Abs), camelids possess Abs consisting of only heavy chains. The variable domain of such a heavy-chain Ab (VHH) is fully capable of antigen (Ag) binding. Earlier analysis of 47 VHHs showed sequence features unique to VHH domains. These include the presence of characteristic amino acid substitutions in positions which, in conventional VH domains are involved in interdomain interactions, and the presence of a long third complementarity-determining region (CDR3) which is frequently constrained by an interloop disulphide bond. Here, we describe a large (152) set of Lama glama VHH cDNAs. Based on amino acid sequence similarity, these and other published camelid VHHs were classified into four subfamilies. Three subfamilies are absent in dromedaries, which have been the primary source of VHHs thus far. Comparison of these subfamilies to conventional VH regions reveals new features characteristic of VHHs and shows that many features earlier regarded as characteristic of VHHs in general are actually subfamily specific. A long CDR3 with a concomitant putative additional disulphide bond is only observed in two VHH subfamilies. Furthermore, we identified new VHH-characteristic residues at positions forming interdomain sites in conventional VH domains. The VHH subfamilies also differ from each other and conventional VH domains in the canonical structure of CDR1 and CDR2, mean CDR3 length, and amino acid residue variability. Since different VHH-characteristic residues are observed in all four subfamilies, these subfamilies must have evolved independently from classical VH domains. (C) 2001 Elsevier Science Ltd. All rights reserved.