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Abstract: The bird spider Ornithoctonus huwena is one of the most venomous spiders in China. Its venom has been investigated but usually only the most abundant components have been analyzed. To characterize the primary structure of O. huwena toxins, a list of transcripts within the venom gland were made using the expressed sequence tag (EST) strategy. We generated 468 ESTs from a directional cDNA library of O. huwena venom glands. All ESTs were grouped into 24 clusters and 65 singletons, of which 68.00% of total ESTs belong to toxin-like sequences, 13.00% are similar to body peptide transcripts and 19.00% have no significant similarity to any known sequences. Precursors of all toxin-like sequences can be classified into eight different superfamilies (HWTX-I superfamily, HWTX-II superfamily, HWTX-X superfamily, HWTX-XIV superfamily, HWTX-XV superfamily, HWTX-XVI superfamily, HWTX-XVII superfamily, HWTX-XVIII superfamily) except HWTX-XI and HWTX-XIII, according to the identity of their precursor sequences. The results have predictive value for the discovery of various groups of pharmacologically distinct toxins in complex venoms, and for understanding the relationship of spider toxin evolution based on the diversification of cDNA sequences, primary structure of precursor peptides, three-dimensional structure motifs and biological functions. [Copyright &y& Elsevier]