Tegumental proteins form a membrane-bound outer surface and are thus involved in host-parasite interactions and parasite survival. A complementary DNA clone encoding a novel 21.6-kDa tegumental protein (CsTegu21.6, accession number JF911532) was identified in a sequence library for the adult Clonorchis sinensis liver fluke. The complete coding sequence was 564 bp and encoded a protein of 188 amino acids. A BLASTX search revealed identities from 43 to 47% with previously identified tegumental proteins in C. sinensis and other helminthic parasites. Multiple alignment of the amino acids of CsTegu21.6 with those of four other C. sinensis tegumental proteins, CsTegu21.1, CsTegu22.3, CsTegu20.8 and CsTegu31.8, revealed pair-wise sequence identities ranging from 24 to 31.8%. A calcium-binding EF-hand domain containing a basic helix-loop-helix structure at the N terminus and a dynein light chain domain at the C terminus were found in CsTegu21.6; these motifs are common in tegumental proteins. CsTegu21.6 was specifically observed on the tegument of adult worms using immunolocalization analysis.