Expressed sequence tag (EST) data representing transcripts with a high level of differential hybridization in suppressive-subtractive hybridization (SSH)-based microarray analysis between adult female and male Ascaris suum were subjected to detailed bioinformatic analysis. A total of 361 ESTs clustered into 209 sequences, of which 52 and 157 represented transcripts that were enriched in female and male A. suum, respectively. Thirty (57.7%) of the ‘female’ subset of 52 sequences had orthologues/homologues in other parasitic nematodes and/or Caenorhabditis elegans, 13 (25%) exclusively in other parasitic nematodes and nine (17.3%) had no match in any other organism for which sequence data are currently available; the C. elegans orthologues encoded molecules involved in reproduction as well as embryonic and gamete development, such as vitellogenins and chitin-binding proteins. Of the ‘male’ subset of 157 sequences, 73 (46.5%) had orthologues/homologues in other parasitic nematodes and/or C. elegans, 57 (37.5%) in other parasitic nematodes only, and 22 (14.5%) had no significant similarity match in any other organism; the C. elegans orthologues encoded predominantly major sperm proteins (MSPs), kinases and phosphatases, actins, myosins and an Ancylostoma secreted protein-like molecule. The findings of the present study should support further genomic investigations of A. suum.